CD133-binding agents and uses thereof

ABSTRACT

This disclosure is directed to novel CD133-binding agents. The disclosure is also directed to uses of novel CD133-binding agents for detecting CD133-expressing cells and/or quantitating levels of cellular CD133 expression, for targeting CD133-expressing cells, for decreasing levels of CD133 in CD133-expressing cells and for treating or preventing cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry of PCT/CA2017/051245 filedOct. 19, 2017 (which designates the U.S.), which claims the benefit ofpriority to U.S. Provisional Application No. 62/410,162 filed Oct. 19,2016, and U.S. Provisional Application No. 62/472,209 filed Mar. 16,2017, the contents of both of which are incorporated herein by referencein their entirety.

Incorporation of Sequence Listing

A computer readable form of the Sequence Listing“25301-P51681US02_SequenceListing.txt” (65,536 bytes), submitted viaEFS-WEB and created on Apr. 17, 2019, is herein incorporated byreference.

FIELD

This disclosure relates generally to CD133-binding agents, and tomethods and uses of these binding agents.

BACKGROUND

CD133 has been identified as a marker in melanoma, brain tumors andvarious carcinomas, including breast, colon, gastric, prostate, liver,pancreatic, lung cancer and head and neck squamous cell carcinoma (Bomanet al., 2008; Ferrandina et al., 2009). CD133 expression is oftenassociated with poor survival, drug resistance and metastasis. Acorrelation between CD133 overexpression, histopathological factors andpoor patient outcome has been reported in hepatocellular carcinoma(Zhong et al., 2015). CD133 is a membrane-bound pentaspan glycoprotein,the exact physiologic role of which remains unclear. It is thought to beinvolved in primitive cell differentiation and epidermal-mesenchymalinteraction (Bauer et al., 2008; Ulasov et al., 2011; Evangelista etal., 2006), and to be associated with the WNT signaling pathway andassociated cell proliferation (Rappa et al., 2008; Mak et al., 2012a;Takenobu et al., 2011). Downregulation of CD133 in a metastatic melanomacell line has been shown to result in reduced metastatic capacity ofxenografts (Rappa et al., 2008).

Glioblastoma (GBM) is a uniformly fatal primary brain tumor,characterized by a diverse cellular phenotype and genetic heterogeneity.Despite the use of aggressive cellular multi-modal treatment includingsurgical resection, radiotherapy and chemotherapy, the outcome ofpatients with GBM has failed to improve significantly. Numerous studieshave implicated CD133+ brain tumor initiating cells (BTICs) as driversof chemo- and radio-resistance in GBM. It has also recently beendemonstrated that a CD133-driven gene signature is predictive of pooroverall survival (Venugopal et al, 2015) and targeting CD133+ treatmentrefractory cells may be an effective strategy to block GBM recurrence.Medulloblastoma cells that are CD133+ have also been associated withincreased multipotency and enriched brain cancer stem cell activity(Singh et al, 2004).

Anti-CD133 antibody-based drugs have been proposed for treatment ofcancer (reviewed in Schmohl and Vallera, 2016). Significant buttemporary regression of recurrent glioblastoma after anti-IL13alphachimeric antigen receptor T-cell therapy has been reported in a humanpatient (Brown et al., 2016). A need remains for novel agents that bindCD133 with high affinity and specificity.

SUMMARY

The present inventors have described novel antibody variable regionsRW01 and RW03 capable of specifically binding both cellsurface-expressed as well as denatured human CD133, and demonstratedspecific CD133-binding by binding agents (e.g. antibodies, Fabs, scFvs,Fab-based bispecific antibodies/bispecific T cell engagers (BiTEs)and/or scFab-based bispecific antibodies/BiTEs) comprising theseCD133-binding variable regions. It was shown that antibodies with thepresently disclosed variable regions specifically bind cellsurface-expressed/native human CD133 with a dissociation constant(K_(D)) in the subnanomolar/nanomolar range. It was shown that suchCD133-binding antibodies can be used to specifically detect CD133expressed on the surface of cells, such as cancer cells (e.g. pancreaticcancer cells, colorectal cancer cells); specifically bind and detectdenatured CD133, e.g. in cell lysates; specifically bind, detect andsubcellularly localize cellular CD133 via immunofluorescence analysis;and significantly reduce levels of CD133 protein in CD133-positive(CD133+) cancer cells. It was further shown that Fab comprising antibodyvariable region RW01 and Fab comprising antibody variable region RW03 donot compete with IgG RW03 and IgG RW01, respectively, for binding toCD133.

The present inventors have also shown that CD133 specific CAR-T cellsspecifically induce CD133-positive glioblastoma (GBM) cell death andinduce GBM tumor regression in vivo. The inventors have also shown thatCD133-specific BiTEs recruit T cells to CD133+ human GBM cells and causecell death. In addition, tumors formed in mouse brain intracraniallytreated with CD133-specific BiTEs were less aggressive and invasive.

Accordingly, the present disclosure provides a CD133-binding agent whichspecifically binds cell surface-expressed/native CD133 and whichspecifically binds denatured CD133.

In one embodiment, the CD133-binding agent specifically binds a CD133epitope bound by an antibody comprising: (a) a light chain having theamino acid sequence of SEQ ID NO: 2, and a heavy chain having the aminoacid sequence of SEQ ID NO: 3; and/or (b) a light chain having the aminoacid sequence of SEQ ID NO: 4 and a heavy chain having the amino acidsequence of SEQ ID NO: 5.

In another embodiment, the CD133-binding agent specifically binds aCD133 epitope of cell surface-expressed CD133 which is bound by anantibody comprising a light chain having the amino acid sequence of SEQID NO: 2 and a heavy chain having the amino acid sequence of SEQ ID NO:3, and/or which is bound by an antibody comprising a light chain havingthe amino acid sequence of SEQ ID NO: 4 and a heavy chain having theamino acid sequence of SEQ ID NO: 5.

In a further embodiment, the CD133-binding agent specifically binds aCD133 epitope of denatured CD133 which is bound by an antibodycomprising a light chain having the amino acid sequence of SEQ ID NO: 2and a heavy chain having the amino acid sequence of SEQ ID NO: 3, and/orwhich is bound by an antibody comprising a light chain having the aminoacid sequence of SEQ ID NO: 4 and a heavy chain having the amino acidsequence of SEQ ID NO: 5.

In one embodiment, the CD133-binding agent comprises an antibody lightchain variable domain and an antibody heavy chain variable domain whichform an antigen binding site that specifically binds human CD133.

In another embodiment, the antibody light chain variable domaincomprises a light chain complementarity-determining region (CDR)1comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2comprising the amino acid sequence of SEQ ID NO: 7, and a light chainCDR3 comprising the amino acid sequence of SEQ ID NO: 8; and theantibody heavy chain variable domain comprises a heavy chain CDR1comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chainCDR3 comprising the amino acid sequence of SEQ ID NO: 11, wherein thelight chain variable domain and the heavy chain variable domain form theantigen binding site that binds human CD133. Optionally, the heavy chainvariable domain further comprises a Met residue at position 39, a Serresidue at position 55 and a Tyr residue at position 66.

In a further embodiment, the antibody light chain variable domaincomprises a light chain complementarity-determining region (CDR)1consisting of the amino acid sequence of SEQ ID NO: 6, a light chainCDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and a lightchain CDR3 consisting of the amino acid sequence of SEQ ID NO: 8; andthe antibody heavy chain variable domain comprises a heavy chain CDR1consisting of the amino acid sequence of SEQ ID NO: 9, a heavy chainCDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and a heavychain CDR3 consisting of the amino acid sequence of SEQ ID NO: 11,wherein the light chain variable domain and the heavy chain variabledomain form the antigen binding site that binds human CD133. Optionally,the heavy chain variable domain further comprises a Met residue atposition 39, a Ser residue at position 55 and a Tyr residue at position66.

In another embodiment, the antibody light chain comprises the amino acidsequence of SEQ ID NO: 2 or an amino acid sequence having at least 70%sequence identity to the framework regions of SEQ ID NO: 2.

In another embodiment, the antibody light chain consists of the aminoacid sequence of SEQ ID NO: 2.

In another embodiment, the heavy chain comprises the amino acid sequenceof SEQ ID NO: 3 or an amino acid sequence having at least 70% sequenceidentity to the framework regions of SEQ ID NO: 3.

In another embodiment, the antibody heavy chain consists of the aminoacid sequence of SEQ ID NO: 3.

In another embodiment, the light chain comprises (i) the amino acidsequence of SEQ ID NO: 2 or an amino acid sequence having at least 70%sequence identity to the framework regions of SEQ ID NO: 2, and (ii) theheavy chain comprises the amino acid sequence of SEQ ID NO: 3 or anamino acid sequence having at least 70% sequence identity to theframework regions of SEQ ID NO: 3.

In another embodiment, the light chain consists of the amino acidsequence of SEQ ID NO: 2, and the heavy chain consists of the amino acidsequence of SEQ ID NO: 3.

In another embodiment, the antibody light chain variable domaincomprises a light chain complementarity-determining region (CDR)1comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2comprising the amino acid sequence of SEQ ID NO: 13, and a light chainCDR3 comprising the amino acid sequence of SEQ ID NO: 14; and theantibody heavy chain variable domain comprises a heavy chain CDR1comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 16, and a heavy chainCDR3 comprising the amino acid sequence of SEQ ID NO: 17, wherein thelight chain variable domain and the heavy chain variable domain form theantigen binding site that binds human CD133. Optionally, the heavy chainvariable domain further comprises a lile residue at position 39, a Tyrresidue at position 55 and a Tyr residue at position 66.

In one embodiment, the antibody light chain variable domain comprises alight chain complementarity-determining region (CDR)1 consisting of theamino acid sequence of SEQ ID NO: 12, a light chain CDR2 consisting ofthe amino acid sequence of SEQ ID NO: 13, and a light chain CDR3consisting of the amino acid sequence of SEQ ID NO: 14; and the antibodyheavy chain variable domain comprises a heavy chain CDR1 consisting ofthe amino acid sequence of SEQ ID NO: 15, a heavy chain CDR2 consistingof the amino acid sequence of SEQ ID NO: 16, and a heavy chain CDR3consisting of the amino acid sequence of SEQ ID NO: 17, wherein thelight chain variable domain and the heavy chain variable domain form theantigen binding site that binds human CD133. Optionally, the heavy chainvariable domain further comprises a Ile residue at position 39, a Tyrresidue at position 55 and a Tyr residue at position 66.

In another embodiment, the antibody light chain comprises the amino acidsequence of SEQ ID NO: 4 or an amino acid sequence having at least 70%sequence identity to the framework regions of SEQ ID NO: 4.

In another embodiment, the antibody light chain consists of the aminoacid sequence of SEQ ID NO: 4.

In another embodiment, the antibody heavy chain comprises the amino acidsequence of SEQ ID NO: 5 or an amino acid sequence having at least 70%sequence identity to the framework regions of SEQ ID NO: 5.

In another embodiment, the antibody heavy chain consists of the aminoacid sequence of SEQ ID NO: 5.

In another embodiment, the light chain comprises the amino acid sequenceof SEQ ID NO: 4 or an amino acid sequence having at least 70% sequenceidentity to the framework regions of SEQ ID NO: 4, and the heavy chaincomprises the amino acid sequence of SEQ ID NO: 5 or an amino acidsequence having at least 70% sequence identity to the framework regionsof SEQ ID NO: 5.

In another embodiment, the light chain consists of the amino acidsequence of SEQ ID NO: 4, and the heavy chain consists of the amino acidsequence of SEQ ID NO: 5.

In another embodiment, the CD133-binding agent is selected from thegroup consisting of an antibody, an antibody fragment, a single-chain Fv(scFv), a bispecific antibody, a phage-Fab wherein the Fab binds CD133and a phage-scFv wherein the scFv binds CD133.

In another embodiment, the CD133-binding agent comprises an antibodythat binds human CD133.

In another embodiment, the CD133-binding agent comprises an antibodyfragment that binds human CD133.

In another embodiment, the CD133-binding agent comprises a single-chainFv (scFv) that binds human CD133.

In another embodiment, the CD133-binding agent comprises a bispecificantibody that binds human CD133.

In another embodiment, the CD133-binding agent comprises a phage-Fab,wherein the Fab binds human CD133.

In another embodiment, the CD133-binding agent comprises a phage-scFvthat binds human CD133, wherein the scFv binds human CD133.

In still another embodiment, the antibody fragment is a fragmentantigen-binding (Fab).

In another embodiment, the CD133-binding agent is (a) a bispecificantibody comprising a CD133-binding single-chain Fab and anon-CD133-binding scFv, (b) a bispecific antibody comprising aCD133-binding Fab and a non-CD133-binding scFv, or (c) a CD133-bindingand CD3-binding bispecific antibody.

In yet another embodiment, the CD133-binding agent is (a) a bispecificantibody comprising a CD133-binding single-chain Fab and a CD3-bindingscFv or (b) a bispecific antibody comprising a CD133-binding Fab and aCD3-binding scFv.

In another embodiment, the CD133-binding agent is a chimeric antigenreceptor (CAR) comprising (i) a CD133-binding antibody variable regionand (ii) a CAR signaling domain comprising one or more immune cellreceptor signaling domains.

In one embodiment, the CD133-binding agent comprises human antibodyconstant regions.

In another embodiment, the CD133-binding agent is an IgG molecule.

In a further embodiment, the IgG molecule is an IgG1 molecule.

In another embodiment, the CD133-binding agent is labelled with adetection agent.

The disclosure also provides an immunoconjugate comprising (1) thebinding agent described above attached to (2) an effector agent.Optionally, the effector agent is an anti-neoplastic agent or a toxin.

The disclosure also provides a pharmaceutical composition comprising theCD133-binding agent or the immunoconjugate described above and acarrier.

The disclosure also provides a use of the CD133-binding agent,immunoconjugate or the pharmaceutical composition described above fortargeting CD133-expressing cells.

The disclosure also provides a use of the CD133-binding agent,immunoconjugate or the pharmaceutical composition described above forbinding CD133-expressing cells.

The disclosure further provides a use of the CD133-binding agent,immunoconjugate or the pharmaceutical composition described above fordetecting CD133-expressing cells and/or quantitating levels of cellularCD133 expression.

The disclosure additionally provides a use of the CD133-binding agent orthe pharmaceutical composition described herein for reducing levels ofCD133 protein in CD133-expressing cells.

In one embodiment, the use of the CD133-binding agent is for detectingand/or quantitating levels of cell-surface expressed CD133 in cells. Inanother embodiment, the use of the CD133-binding agent is for detectingand/or quantitating total levels of CD133 in cells.

Optionally, detecting CD133-expressing cells and/or quantitating levelsof cellular CD133 expression is done by Western blotting, enzyme linkedimmunosorbent assay (ELISA), immunofluorescence, immunohistochemistry orflow cytometry.

In another embodiment, the cells are cancer cells, optionallyCD133-expressing cancer cells or cancer cells detectably expressingCD133.

In yet another embodiment, the cancer cells are melanoma cancer cells,pancreatic cancer cells, brain cancer cells or colorectal cancer cells.In another embodiment, the cancer cells are glioblastoma cells. Inanother embodiment, the cancer cells are medulloblastoma cells.

The disclosure additionally provides use of a CD133-binding agent,immunoconjugate or the pharmaceutical composition described herein fortreating or preventing a cancer.

In one embodiment, the cancer is a CD133-expressing cancer or a cancerdetectably expressing CD133. In another embodiment, the cancer ismetastatic melanoma, brain, prostate, pancreatic or colon cancer.

In another embodiment, the brain cancer is a glioblastoma, optionally aCD133-expressing glioblastoma or a glioblastoma detectably expressingCD133. In another embodiment, the brain cancer is a medulloblastoma,optionally a CD133-expressing medulloblastoma or a medulloblastomadetectably expressing CD133.

The disclosure also provides a use of a CD133-binding agent comprising(a) a CD133-binding single-chain Fab and a non-CD133-binding scFv, (b) abispecific antibody comprising a CD133-binding Fab and anon-CD133-binding scFv, or (c) a CD133-binding and CD3-bindingbispecific antibody, for treating glioblastoma, optionallyCD133-expressing glioblastoma or glioblastoma detectably expressingCD133.

The disclosure also provides a use of a CD133-binding agent comprising(a) a CD133-binding single-chain Fab and a CD3-binding scFv or (b) abispecific antibody comprising a CD133-binding Fab and a CD3-bindingscFv for treating glioblastoma, optionally CD133-expressing glioblastomaor glioblastoma detectably expressing CD133.

The disclosure also provides a use of a CD133-binding agent comprising(a) a CD133-binding single-chain Fab and a non-CD133-binding scFv, (b) abispecific antibody comprising a CD133-binding Fab and anon-CD133-binding scFv, or (c) a CD133-binding and CD3-bindingbispecific antibody, for treating medulloblastoma, optionallyCD133-expressing medulloblastoma or medulloblastoma detectablyexpressing CD133.

The disclosure also provides a use of a CD133-binding agent comprising(a) a CD133-binding single-chain Fab and a CD3-binding scFv or (b) abispecific antibody comprising a CD133-binding Fab and a CD3-bindingscFv for treating medulloblastoma, optionally CD133-expressingmedulloblastoma or medulloblastoma detectably expressing CD133.

In one embodiment, the bispecific antibody comprises an amino acidsequence comprising:

(a) SEQ ID NO: 22 and SEQ ID NO: 23,

(b) SEQ ID NO: 24 and SEQ ID NO: 25,

(c) SEQ ID NO: 26,

(d) SEQ ID NO: 27, or functional variants thereof.

The disclosure also provides a use of a T-cell expressing a chimericantigen receptor (CAR) described herein for treating glioblastoma,optionally CD133-expressing glioblastoma or glioblastoma detectablyexpressing CD133.

The disclosure further provides a use of a T-cell expressing a chimericantigen receptor (CAR) described herein for treating medulloblastoma,optionally CD133-expressing medulloblastoma or medulloblastomadetectably expressing CD133.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating embodiments of the disclosure are given by wayof illustration only, since various changes and modifications within thespirit and scope of the disclosure will become apparent to those skilledin the art from this detailed description.

DRAWINGS

Embodiments are described below in relation to the drawings in which:

FIG. 1A is a table depicting the results of a cell-based ELISA forCD133-binding of phage-Fab clones and phage-scFv clones selected fromLibrary F or Library G, respectively, for binding to cell-surface CD133using the Cellectseq method. Following cell selections, round fouroutput phage for each library was plated for single colonies. Thesecolonies were grown up in an overnight culture and tested for binding tocells by cell-based ELISA. The plates were read and OD450 nm wasdetected and recorded. The assay measured binding of clones toCD133-overexpressing HEK293-CD133 cells (under “CD133” column headers)vs parental HEK293 cells (under “HEK293” column headers).

FIG. 1B is a histogram depicting a cell-based ELISA for CD133-binding ofthe three clones (phage-Fab RW03, phage-Fab C12 and phage-Fab F5) whichwere selected from Library F for binding to cell-surface CD133 using theCellectseq method, and which were found, after DNA sequencing, torepresent the three unique antibody variable regions present in 77clones found to preferentially bind to HEK293-CD133 cells vs HEK293cells by at least 1.5-fold.

FIG. 2 is a series of fluorescence photomicrographs depicting thatphage-Fab clone RW03 specifically binds to CD133 overexpressing cells.The three clones with unique sequences obtained from the Library Fcell-based ELISA were used as probes in an immunofluorescence assay.Phage-Fab clones C12 and F5 are shown to bind to HEK293 cellsnon-specifically whereas the RW03 clone binds to HEK293-CD133 cellsspecifically with little background binding to HEK293 cells.

FIG. 3A and FIG. 3B are a histogram and a set of fluorescencephotomicrographs, respectively, depicting that purified Fab RW03specifically binds to HEK293-CD133 as opposed to HEK293 cells. Expressedand purified Fab RW03 was tested for binding by cell-based ELISA (FIG.3A) and immunofluorescence (IF) assay (FIG. 3B).

FIG. 4A and FIG. 4B are line graphs depicting binding curves for bindingof IgG RW01 and IgG RW03, respectively, to HEK293-CD133 cells, forestimation of binding affinity (EC50). Cells were incubated withstepwise dilutions of either IgG RW01 or IgG RW03 to determine ahalf-maximal binding curve for the antibodies. Using the SigmaPlotgraphing software the EC50 for IgG RW01 was calculated as 2.5 nM (FIG.4A) and the EC50 for IgG RW03 was calculated as 0.5 nM (FIG. 4B).

FIG. 5 is a set of fluorescence histograms depicting that IgG RW01 andIgG RW03 can be used to specifically bind and detect cell surface CD133in pancreatic cancer cells and colorectal cancer cells, as shown viaflow cytometry analysis.

FIG. 6 is a set of fluorescence photomicrographs depicting that IgG RW01and IgG RW03 can be used to specifically bind, detect and subcellularlylocalize cellular CD133, as shown via immunofluorescence analysis. Theantibodies were tested for binding to HEK293-CD133 and HEK293 cells.

FIG. 7 is a set of photographs of a Western blot analysis depicting thatIgG RW01 and IgG RW03 can be used to detect denatured CD133/cellularCD133 in colorectal cancer cells, as shown via Western blot analysis.Whole cell lysates of HEK293, HEK293-CD133 and Caco-2 cells were probedwith IgG RW01 and IgG RW03 and binding was detected with an anti-humanHRP-conjugated secondary antibody. Beta-actin was used as a loadingcontrol.

FIG. 8 shows that Fab comprising antibody variable region RW01 and Fabcomprising antibody variable region RW03 do not compete with IgG RW03and IgG RW01, respectively, for binding to CD133. RW01 and RW03 weretested for binding to CD133 in a competitive flow cytometry experiment.In (a) cells were incubated with stepwise dilutions of IgG RW01(circles) or IgG RW01 in the presence of Fab RW03 (squares). Similarly,in (b) cells were incubated stepwise with dilutions of IgG RW03(circles) or IgG RW03 in the presence of Fab RW01 (squares).

FIG. 9 is a set of photographs of a Western blot analysis depicting thattreatment with IgG RW01 or IgG RW03 significantly reduces total cellularlevels of CD133 protein in Caco-2 colorectal cancer cells. Caco-2 cellswere incubated with the indicated antibody for 24-hours at 37° C., wholecell lysates were prepared and probed with AC133 anti-CD133 antibody.Anti-human IgG (H+L) antibody was used as negative antibody control, andGAPDH was used as a loading control.

FIG. 10A is a set of schematic diagrams depicting the configurations ofBiTE #1, BiTE #2, BiTE #3 and BiTE #4.

FIG. 10B is a photograph of a Western blot analysis depicting that BiTE#1, BiTE #2, BiTE #3 and BiTE #4 could each be expressed and purifiedfrom HEK293 cells by transient transfection protocol.

FIG. 11A and FIG. 11B are sets of fluorescence histograms depicting thatBiTE #1 (FIG. 10A), BiTE #2 (FIG. 10A), BiTE #3 (FIG. 10B) and BiTE #4(FIG. 10B) each binds to HEK293-CD133 cells significantly more than tothe parental HEK293 cells, even at concentrations as low as 0.073-0.11microgram/ml, as determined via flow cytometry. The BiTE concentrationsemployed are indicated for each histogram. In each histogram therightmost peak represents binding to HEK293-CD133 cells and the leftmostpeak represents binding to the parental HEK293 cells.

FIG. 12 is a histogram of ELISA results depicting that BiTE #1, BiTE #2,BiTE #3 and BiTE #4 each binds to CD3 in the form of CD3 epsilon/gammaand CD3 epsilon/delta. Anti-CD3 antibodies UCHT1 and OKT3 were used aspositive antibody controls and BSA was used as no antibody control.

FIG. 13 depicts constructs for the generation of CD133-specific chimericantigen receptors (CARs).

FIG. 14 shows the characterization of CAR-T cells.

FIG. 15 shows the validation of CD133-specific CAR-T cells.

FIG. 16 shows that CD133-specific CAR-T cells are activated in presenceof CD133+ human GBM cells.

FIG. 17 shows that activated CD133-specific CAR-T cells have enhancedproliferation ability and specifically induce CD133-positive GBM celldeath and CD133-positive medulloblastoma cell death. CAR-T cells wereco-cultured with CD133^(high) and CD133^(low) GBM cells as well asCD133^(high) medulloblastoma cells. Flow cytometry was based onlive-dead staining with IR-dye.

FIG. 18A-C shows that CD133-specific T cells induce GBM tumor regressionin vivo. Treatment was delivered intracranially at a dose of 1 million×2doses (2 week).

FIGS. 19A and B shows the development of CD133×CD3 BiTEs.

FIGS. 20A and B shows that CD133×CD3 BiTEs bind to CD133+ GBM tumorcells and CD3+T lymphocytes.

FIG. 21 shows that CD133-specific BiTEs activate T cells.

FIG. 22A-C shows that CD133-specific BiTEs recruit T cells to CD133+human GBM cells and cause cell death.

FIG. 23A-D shows a CD133×CD3 BiTE mediated antitumor response.

DESCRIPTION OF VARIOUS EMBODIMENTS

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. For example,the term “a cell” includes a single cell as well as a plurality orpopulation of cells. Generally, nomenclatures utilized in connectionwith, and techniques of, cell and tissue culture, molecular biology, andprotein and oligonucleotide or polynucleotide chemistry andhybridization described herein are those well-known and commonly used inthe art (see, e.g. Green and Sambrook, 2012).

Terms of degree such as “about”, “substantially”, and “approximately” asused herein mean a reasonable amount of deviation of the modified termsuch that the end result is not significantly changed. These terms ofdegree should be construed as including a deviation of at least ±5% ofthe modified term if this deviation would not negate the meaning of theword it modifies.

Compositions of Matter:

The present inventors have provided novel synthetic antibody variableregions which are capable of specifically binding surfaceexpressed/native human CD133 and also denatured human CD133, and whichspecifically bind human CD133 with a dissociation constant (K_(D)) inthe subnanomolar/nanomolar range (see Examples 3 and 6, below).

The inventors have particularly provided CD133-binding phage-scFv cloneRW01 wherein the scFv comprises CD133-binding antibody variable regionRW01, and CD133-binding phage-Fab clone RW03 wherein the Fab comprisesCD133-binding antibody variable region RW03. These clones were selectedfrom phage display libraries for their capacity to specifically bindCD133-expressing cells, as initially confirmed via enzyme-linkedimmunosorbent assay (ELISA; see Examples 1 and 2, below). The inventorshave further particularly provided that CD133-binding antibody variableregion RW01 comprises an antibody light chain variable domaincorresponding to the Asp1 to Lys106 segment of SEQ ID NO: 2, wherein theamino acid sequences of the light chain CDR1, light chain CDR2 and lightchain CDR3 thereof correspond to SEQ ID NO: 6, SEQ ID NO: 7, and SEQ IDNO: 8, respectively (see Example 2, below). The inventors haveadditionally provided that antibody variable region RW01 comprises anantibody heavy chain variable domain corresponding to the Glu1 to Thr120segment of SEQ ID NO: 3, wherein the amino acid sequences of the heavychain CDR1, heavy chain CDR2 and heavy chain CDR3 thereof correspond toSEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, andwherein the (framework region) residues at positions 39, 55 and 66 areMet, Ser and Tyr residues (see Example 2, below).

The inventors have yet further provided that CD133-binding antibodyvariable region RW03 comprises an antibody light chain variable domaincorresponding to the Asp1 to Lys109 segment of SEQ ID NO: 4, wherein theamino acid sequences of the light chain CDR1, light chain CDR2 and lightchain CDR3 thereof correspond to SEQ ID NO: 12, SEQ ID NO: 13, and SEQID NO: 14, respectively (see Example 2, below). The inventors have stillfurther provided that antibody variable region RW03 comprises anantibody heavy chain variable domain corresponding to the Glu1 to Ser118segment of SEQ ID NO: 5; wherein the amino acid sequences of the heavychain CDR1, heavy chain CDR2 and heavy chain CDR3 thereof correspond toSEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; andwherein the (framework region) residues at positions 39, 55 and 66 areIle, Tyr and Tyr residues, respectively; (see Example 2, below).

The inventors have further particularly provided that IgG1 antibodies“IgG RW01” and “IgG RW03”, which comprise CD133-binding antibodyvariable region RW01 and CD133-binding antibody variable region RW03,respectively, can be used to: (i) specifically bind and detect cellsurface CD133 in pancreatic cancer cell lines and colorectal cancer celllines, as shown via flow cytometry analysis (see Example 4, below); (ii)specifically bind, detect and subcellularly localize cellular CD133 inCD133-expressing cells, as shown via immunofluorescence analysis (seeExample 5, below); (iii) detect denatured CD133 in whole cell lysate ofcolorectal cancer cells, as shown via Western blot analysis (see Example6, below); and (iv) significantly reduce total cellular CD133 proteinlevels in a colorectal cancer cell line (see Example 8, below). Theinventors disclose that antibody IgG RW01 comprises a light chain havingthe amino acid sequence of SEQ ID NO: 2 and a heavy chain having theamino acid sequence of SEQ ID NO: 3, and antibody IgG RW03 comprises alight chain having the amino acid sequence of SEQ ID NO: 4 and a heavychain having the amino acid sequence of SEQ ID NO: 5 (Example 2). Theinventors have still further disclosed that Fab comprising antibodyvariable region RW01 and Fab comprising antibody variable region RW03 donot compete with IgG RW03 and IgG RW01, respectively, for binding toCD133 (Example 7, below).

The present inventors have also described a chimeric antigen receptor(CAR) T-cell-based strategy that specifically targets CD133+ GBM cells.CD133-specific CAR-expressing T cells were activated in presence ofCD133^(high) GBM cells, and showed increased surface expression ofactivation markers CD69 and CD25. Both, CD4+ and CD8+CD133-specificCAR-T cells showed upregulation in surface expression levels ofactivation markers. The inventors further demonstrated CAR-Tcell-induced cytotoxicity against treatment-resistant and evasive CD133+GBM BTICs (Example 10, below).

The inventors have further particularly disclosed that multipleconfigurations of a bispecific antibody/bispecific T cell engager (BiTE)comprising a scFv which binds the T cell coreceptor CD3, and furthercomprising a Fab or single-chain Fab (scFab) incorporating CD133-bindingantibody variable region RW03, can specifically bind both CD133-positivecells and CD3 (see Example 9, below). The inventors also showed that arecombinant CD133×CD3 bispecific T-cell engager (BiTE) redirects humanpolyclonal T cells to CD133+ GBM cells, inducing a potent anti-tumorresponse (see Example 11, below).

The CD133 molecule is a transmembrane protein having an extracellularN-terminal region, five transmembrane domains with alternating short andlong intracellular and extracellular domains, respectively, and anintracellular C-terminal region. As used herein, CD133 may be from anyspecies or source and includes isoforms, analogs, variants or functionalderivatives of such a CD133 protein. In one embodiment, CD133 is humanCD133. The human CD133 gene or protein may have any of the knownpublished sequences for CD133 which can be obtained from public sourcessuch as GenBank. An example of such a protein sequence includes, but isnot limited to the sequence set out as SEQ ID NO: 1. Human CD133 isalternately referred to in the art as Prominin-1.

CD133-Binding Agents

Accordingly, the disclosure provides a CD133-binding agent whichspecifically binds cell surface-expressed/native CD133 and whichspecifically binds denatured CD133.

As used herein, a CD133-binding agent which “specifically binds cellsurface-expressed/native CD133” is an agent which binds CD133-expressingcells. Cells expressing CD133 can be identified as such, e.g. via flowcytometric analysis, e.g. as described in Example 4. Alternately, aCD133-binding agent which “specifically binds cellsurface-expressed/native CD133” is an agent which binds CD133-expressingcells expressing CD133 at undetectable levels, e.g. at levels below thelimit of detection of an assay such flow cytometric analysis. As usedherein, a CD133-binding agent which “specifically binds denatured CD133”is an agent which binds CD133 in a sample of denatured whole cellprotein of CD133-expressing cells as opposed to the other polypeptidesin the sample (as determined, e.g. via Western blot analysis, e.g. asdescribed in Example 6). The terms “immunoreacts with CD133”, or “isdirected against CD133”, or is characterized as “anti-CD133” are alsoused herein for the same purpose.

In one embodiment, the CD133-binding agent specifically binds a CD133epitope bound by an antibody comprising a light chain having the aminoacid sequence of SEQ ID NO: 2 and a heavy chain having the amino acidsequence of SEQ ID NO: 3 (i.e. antibody IgG RW01), and/or specificallybinds a CD133 epitope bound by an antibody comprising a light chainhaving the amino acid sequence of SEQ ID NO: 4 and a heavy chain havingthe amino acid sequence of SEQ ID NO: 5 (i.e. antibody IgG RW03). In anembodiment, the CD133 epitope is a human CD133 epitope.

As used herein, the term “epitope” refers to the specific site orspecific combination of sites/amino acids on an antigen that are boundby antibody IgG RW01 and/or antibody IgG RW03, for example, unmodifiedor modified (e.g. post-translationally modified, e.g. glycosylated)amino acid residues of human CD133, the minimal polypeptide segment ofhuman CD133 encompassing these amino acid residues, or any combinationof polypeptide segments of human CD133 encompassing these amino acidresidues. Epitopic determinants usually consist of molecules such asamino acids or sugar side chains and usually have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics.

As used herein, unless otherwise specified, an antibody or a bivalentantibody fragment (e.g. F(ab′)₂) referred to as comprising “a” specificlight chain or “a” specific heavy chain in the singular refers to anantibody or a bivalent antibody fragment in which both light chains orboth heavy chains are identical, respectively.

Embodiments of the CD133-binding agent include any type of CD133-bindingmolecule, macromolecule, substance, compound, material, composition, orcomplex, without limitation.

In one embodiment, the CD133-binding agent is a polypeptide. In otherembodiments, the CD133-binding agent is a non-polypeptidic agent, suchas a CD133-binding nucleic acid or a CD133-binding organic compound. TheCD133-binding agent may be monomeric or multimeric. The CD133-bindingagent may be polymeric or non-polymeric. Alternately, the CD133-bindingagent may be an engineered polypeptide (e.g. a naturally occurringpolypeptide engineered to have a modified amino acid sequence; or achimeric polypeptide engineered to comprise two or more naturallyoccurring amino acid sequences; or an engineered polypeptide selectedfrom a library of engineered polypeptides having randomized amino acidsequences), or a chemically modified polypeptide.

In one embodiment, the CD133-binding agent comprises a CD133-bindingantibody variable region.

As used herein, a CD133-binding antibody variable region is acombination of an antibody heavy chain variable domain and an antibodylight chain variable domain, where the antibody heavy chain variabledomain and the antibody light chain variable domain form anantigen-binding site that specifically binds CD133.

The CD133-binding agent is optionally an antibody, an antigen-bindingfragment of an antibody, or an agent comprising a CD133-binding antibodyvariable region.

As used herein, and unless otherwise specified, the term “antibody”refers to an immunoglobulin (Ig) molecule. The basic antibody structuralunit is known to comprise a tetramer. Each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one light (“L”)(about 25 kDa) and one heavy (“H”) chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition, and described in more detail below. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function. The term “antigen-binding site” or “bindingportion” refers to the part of the binding protein that participates inantigen binding. In an antibody, the antigen binding site is formed byamino acid residues of the N-terminal variable (“V”) regions of theheavy and light chains. Three highly divergent stretches within the Vregions of the heavy and light chains, referred to as “hypervariableregions”, are interposed between more conserved flanking stretches knownas “framework regions”, or “FRs”. Thus, the term “FR” refers to aminoacid sequences which are naturally found between, and adjacent to,hypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other inthree-dimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs”. All CDRs and frameworkregions (FRs) disclosed herein, amino acid sequences of CDRs and FRsdisclosed herein, and CDR-encoding or FR-encoding nucleic acid sequencesdisclosed herein, are intended to be defined in accordance with IMGTnumbering (Lefranc et al., 2003). Another system alternately employed inthe art for such definitions is that of Kabat numbering (Kabat et al.,1991).

The CD133-binding agent may be an antibody, such as a human antibody,containing engineered variable regions (e.g. containing variable regionsselected from a phage display library displaying engineered antibodyvariable regions, e.g. a phage-Fab library or a phage-scFv library, e.g.as described in Example 1), or a chimeric antibody comprising humanconstant regions and an antibody variable region of a non-human mammal.The CD133-binding agent may be a humanized antibody, e.g. an antibodycomprising human constant regions, human variable region frameworkregions, and CD133-binding CDRs generated in a non-human mammal. Thenon-human mammal may be a rodent, such as a mouse, rat, rabbit, guineapig or hamster. Alternately, the non-human mammal may be an ungulate,such as a camelid or a bovid. The CD133-binding agent may be an antibodycomprising heavy chain constant regions belonging to any type of class,or subclass. The CD133-binding agent may comprise any type of lightchain.

In one embodiment, the CD133-binding agent is a human antibody, such asan IgG1 antibody, wherein the heavy chain constant regions are gamma1heavy chain constant regions. In other embodiments, the CD133-bindingagent is a human antibody, such as an IgA1, IgA2, IgD, IgG2, IgG3, IgG4,IgE or IgM antibody, wherein the heavy chain constant regions arealpha1, alpha2, delta, gamma2, gamma3, gamma4, epsilon or mu heavy chainconstant regions, respectively.

In yet a further embodiment, the CD133-binding agent is an antibodywherein the light chains comprise human kappa light chain constantdomains, or wherein the light chains are human kappa light chains.Alternately, the CD133-binding agent is an antibody wherein the lightchains comprise human lambda light chain constant domains, or whereinthe light chains are human lambda light chains.

In still a further embodiment the CD133-binding agent is an antibodycomprising human gamma1 heavy chain constant regions and human kappalight chains.

Embodiments of CD133-binding agents of the present disclosure furtherinclude, but are not limited to, fragment antigen-binding (Fab),single-chain Fv (scFv), single-chain Fab (scFab), Fab′, Fv, chemicallylinked F(ab′)₂, dsFv, dsFv′, sc(Fv)₂, ds-scFv, (dsFv)2, scFv-Fc,scFv-based chimeric antigen receptors (CARs), Fab-based CARs,scFab-based CARs, single-chain immunoglobulin (e.g. sclgG),single-domain antibody (sdAb, nanobody), scFv-Fc, minibody (scFv-CH3),diabody, tribody, tetrabody, multimeric antibody (e.g. scFv dimer,bivalent diabody), multispecific antibody (e.g. bispecific antibody,trispecific antibody, di-scFv, tri-scFv, bispecific Fab₂, trispecificFab₂, trispecific triabody, trispecific Fab₃), multimeric/multispecificantibody (e.g. scFv dimer, bispecific diabody, dsFv-dsFv′), heavy-chainantibody, Fab₃, divalent VHH, pentavalent VHH (pentabody), (scFv-SA)₄and, [sc(Fv)2]₂.

In another embodiment, the CD133-binding agent is a phage displaying apolypeptide comprising a CD133-binding antibody variable region, such asa phage-Fab or phage-scFv (see Examples 1 and 2, below).

Embodiments of CD133-binding agents of the present disclosure stillfurther include CD133-binding nucleic acid aptamers (e.g. RNA aptamersor DNA aptamers; see, e.g. Lipi et al., 2016), peptide aptamers (see,e.g. Parashar, 2016), and chemically synthesized agents (e.g. syntheticantibody mimics; see, e.g. McEnaney et al., 2014).

In another embodiment, the CD133-binding agent is a peptide analog.Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics” (see, e.g. Fauchere, 1986); Veber andFreidinger, 1985; and Evans et al., 1987). Such compounds are oftendeveloped with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to biologically useful peptidesmay be used to produce an equivalent biological effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH2NH—, —CH₂S—, —CH2-CH2-, —CH═CH— (cis and trans), —COCH2-, CH(OH)CH2-and —CH2SO—, by methods well known in the art. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g. D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (see,e.g. Rizo and Gierasch, 1992), for example, by adding internal cysteineresidues capable of forming intramolecular disulfide bridges whichcyclize the peptide.

In an embodiment, the CD133-binding agent comprises antibody variableregion RW01, which comprises (a) an antibody light chain variable domain(SEQ ID NO: 28) corresponding to the Asp1 to Lys106 segment of SEQ IDNO: 2, wherein the amino acid sequences of the light chain CDR1, lightchain CDR2 and light chain CD3 thereof correspond to SEQ ID NO: 6, SEQID NO: 7, and SEQ ID NO: 8, respectively (see Example 2, below) and (b)an antibody heavy chain variable domain (SEQ ID NO: 29) corresponding tothe Glu1 to Thr120 segment of SEQ ID NO: 3, wherein the amino acidsequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3thereof correspond to SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11,respectively, and wherein the (framework region) residues at positions39, 55 and 66 are Met, Ser and Tyr residues (see Example 2, below).

In a further embodiment, the CD133-binding agent comprises antibodyvariable region RW03, which comprises (a) an antibody light chainvariable domain (SEQ ID NO: 30) corresponding to the Asp1 to Lys109segment of SEQ ID NO: 4, wherein the amino acid sequences of the lightchain CDR1, light chain CDR2 and light chain CD3 thereof correspond toSEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively (seeExample 2, below) and (b) an antibody heavy chain variable domain (SEQID NO: 31) corresponding to the Glu1 to Ser118 segment of SEQ ID NO: 5;wherein the amino acid sequences of the heavy chain CDR1, heavy chainCDR2 and heavy chain CD3 thereof correspond to SEQ ID NO: 15, SEQ ID NO:16, and SEQ ID NO: 17, respectively; and wherein the (framework region)residues at positions 39, 55 and 66 are Ile, Tyr and Tyr residues,respectively (see Example 2, below).

Also particularly disclosed herein is a CD133-binding agent IgG RW01comprising a light chain amino acid sequence as shown in SEQ ID NO: 2and a heavy chain amino acid sequence as shown in SEQ ID NO: 3 (seeExample 2, below).

Yet further particularly disclosed herein is a CD133-binding agent IgGRW03 comprising a light chain amino acid sequence as shown in SEQ ID NO:4 and a heavy chain amino acid sequence as shown in SEQ ID NO: 5 (seeExample 2, below).

Accordingly, the disclosure also provides a CD133-binding agentcomprising:

(i) an antibody light chain variable domain comprising a light chainCDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chainCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and/or a lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and/or

(ii) an antibody heavy chain variable domain comprising a Met residue atposition 39 and a heavy chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 9; a Ser residue at position 55, a Tyr residue at position 66and a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:10; and/or a heavy chain CDR3 comprising the amino acid sequence of SEQID NO: 11, wherein the antibody light chain variable domain and theantibody heavy chain variable domain form an antigen binding site thatbinds human CD133.

In one embodiment, the antibody light chain variable domain comprises alight chain CDR1, a light chain CDR2, and a light chain CDR3 comprisingthe amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8,respectively; and the antibody heavy chain variable domain comprises aheavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprisingthe amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:11, respectively; and the antibody heavy chain variable domain comprisesMet, Ser and Tyr residues at positions 39, 55 and 66, respectively.

In another embodiment, the antibody light chain variable domaincomprises a light chain CDR1, a light chain CDR2, and a light chain CDR3consisting of the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 andSEQ ID NO: 8, respectively; and the antibody heavy chain variable domaincomprises an amino acid sequence (SEQ ID NO: 32) composed of a heavychain CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, and aMet residue flanking the heavy chain CDR1 at position 39; an amino acidsequence (SEQ ID NO: 33) composed of a heavy chain CDR2 consisting ofthe amino acid sequence of SEQ ID NO: 10, a Ser residue flanking theheavy chain CDR2 at position 55, and a Tyr residue flanking the heavychain CDR2 at position 66; and a heavy chain CDR3 consisting of theamino acid sequence of SEQ ID NO: 11.

In a further embodiment, the light chain comprises the amino acidsequence of SEQ ID NO: 2 or an amino acid sequence having at least 70%sequence identity to the framework regions of SEQ ID NO: 2; and/or theheavy chain comprises the amino acid sequence of SEQ ID NO: 3 or anamino acid sequence having at least 70% sequence identity to theframework regions of SEQ ID NO: 3.

In still a further embodiment, the light chain consists of the aminoacid sequence of SEQ ID NO: 2, and/or the heavy chain consists of theamino acid sequence of SEQ ID NO: 3.

Accordingly, the disclosure also provides a CD133-binding agentcomprising:

(i) an antibody light chain variable domain comprising a light chainCDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chainCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and/or a lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and/or

(ii) an antibody heavy chain variable domain comprising a Ile residue atposition 39 and a heavy chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 15; Tyr residues at positions 55 and 66 and a heavy chainCDR2 comprising the amino acid sequence of SEQ ID NO: 16; and/or a heavychain CDR3 comprising the amino acid sequence of SEQ ID NO: 17, whereinthe antibody light chain variable domain and the antibody heavy chainvariable domain form an antigen binding site that binds human CD133.

In one embodiment, the antibody light chain variable domain comprises alight chain CDR1, a light chain CDR2, and a light chain CDR3 comprisingthe amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO:14, respectively; and the antibody heavy chain variable domain comprisesa heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3comprising the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16 andSEQ ID NO: 17, respectively; and the antibody heavy chain variabledomain comprises Ile, Tyr and Tyr residues at positions 39, 55 and 66,respectively.

In another embodiment, the antibody light chain variable domaincomprises a light chain CDR1, a light chain CDR2, and a light chain CDR3consisting of the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13and SEQ ID NO: 14, respectively; and the antibody heavy chain variabledomain comprises an amino acid sequence (SEQ ID NO: 34) composed of aheavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 15and a lile residue flanking the heavy chain CDR1 at position 39; anamino acid sequence (SEQ ID NO: 35) composed of a heavy chain CDR2consisting of the amino acid sequence of SEQ ID NO: 16, a Tyr residueflanking the heavy chain CDR2 at position 55, and a Tyr residue flankingthe heavy chain CDR2 at position 66; and a heavy chain CDR3 consistingof the amino acid sequence of SEQ ID NO: 17.

In a further embodiment, the antibody light chain comprises the aminoacid sequence of SEQ ID NO: 4 or an amino acid sequence having at least70% sequence identity to the framework regions of SEQ ID NO: 4; and/orthe antibody heavy chain comprises the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 70% sequence identity to theframework regions of SEQ ID NO: 5.

In a still further embodiment, the light chain consists of the aminoacid sequence of SEQ ID NO: 4, and the heavy chain consists of the aminoacid sequence of SEQ ID NO: 5.

Any of the CD133-binding agents of the present disclosure may beobtained and suitably prepared for use using well-known techniques.

Polypeptidic CD133-binding agents of the disclosure can be synthesizedby recombinant techniques which are well known and routinely practicedin the art. A polypeptidic CD133-binding agent of the disclosure may beproduced in recombinant sources, such as recombinant cell lines ortransgenic animals. Techniques can be adapted for the production ofsingle-chain antibodies, such as a scFv, specific to CD133 (see, e.g.U.S. Pat. No. 4,946,778).

Alternatively, a polypeptidic CD133-binding agent of the disclosure,such as a CD133-binding antibody of the disclosure may be obtained byimmunizing an animal with CD133, or with a polypeptide comprising asuitable CD133 epitope, so as to generate the antibody in the animal'sserum.

A CD133-binding IgG antibody of the disclosure can be purified from abiological sample, such as serum, via techniques such as affinitychromatography using protein A or protein G (see, e.g. Wilkinson, 2000).Additionally or alternatively, CD133, or a polypeptide comprising anepitope thereof, which is specifically bound by the CD133-binding agentmay be immobilized on a column to purify the CD133-binding agent from asample by immunoaffinity chromatography.

A CD133-binding antibody fragment of the disclosure may be obtained froman antibody using conventional techniques. For example, F(ab′)2fragments can be generated by treating an antibody with pepsin. Theresulting F(ab′)2 fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments.

Methods of producing polypeptidic CD133-binding agents of the disclosureare described in further detail below.

As set forth above, in an embodiment, the CD133-binding agent may be abispecific antibody.

As used herein, bispecific antibodies are binding agents comprising twodifferent antibody variable regions which confer binding specificitiesfor at least two different antigens or two different epitopes of thesame antigen.

The presently disclosed bispecific antibodies specifically bind CD133and another antigen or specifically bind different epitopes of CD133.Optionally, the bispecific antibody binds CD133 and a cell-surfaceprotein, receptor or receptor subunit.

In one embodiment, the bispecific antibody comprises a CD133-bindingsingle-chain Fab and a non-CD133-binding scFv. Alternately, thebispecific antibody comprises a CD133-binding Fab and anon-CD133-binding scFv.

In another embodiment, the CD133-binding agent is a bispecific antibodythat targets, binds and/or engages immune cells such as T cells,macrophages or NK cells. According to this embodiment, the CD133-bindingagent is a bispecific antibody where one of the binding specificities isfor CD133 and the other binding specificity is for an antigen expressedon the surface of T cells, macrophages or NK cells. For example, thebispecific antibody may bind CD133 and an immune cell receptor, such areceptor of a T cell, which when bound activates or inhibits activity ofthe immune cell.

Various techniques for making and isolating bispecific antibodiesdirectly from recombinant cell culture have been described. For example,bispecific antibodies have been produced using leucine zippers (see,e.g. Kostelny et al., 1992), using “diabody” technology (see, e.g.Hollinger et al., 1993), and using single-chain Fv (scFv) dimers (see,e.g. Gruber et al., 1994).

A bispecific antibody that engages T cells may be referred to as abispecific T-cell engager (BiTE). In one embodiment of the presentdisclosure, the bispecific antibody/BiTE specifically binds both CD133and the T cell co-receptor CD3 (also referred to herein asCD133-binding/CD3-binding bispecific antibody). Accordingly, providedherein is a bispecific antibody/BiTE which comprises a CD133-bindingantibody variable region of the disclosure and a CD3-binding antibodyvariable region. Such bispecific antibodies/BiTEs allow targeting of a Tcell to a cell, such as a cancer cell, expressing surface CD133. Variousconfigurations of the bispecific antibodies/BiTEs are contemplatedherein. For example, in one embodiment, the bispecific antibody/BiTEcomprises an anti-CD133 Fab and an anti-CD3 scFv. Optionally, either thelight chain or the heavy chain of the anti-CD133 Fab is linked to theheavy-chain of the anti-CD3 scFv. In another embodiment, the bispecificantibody/BiTE comprises an anti-CD133 single chain Fab (scFab) and ananti-CD3 ScFv. Optionally, either the light chain or the heavy chain ofthe anti-CD133 Fab or anti-CD133 scFab is linked to the heavy-chain ofthe anti-CD3 scFv. In one embodiment, the anti-CD3 scFv binds CD3epsilon/gamma. In one embodiment, the BiTE/bispecific antibody binds CD3epsilon/delta. See, for example FIG. 10A. Examples of configurations andamino acid sequences of various embodiments of the BiTE are provided inTable 6. Accordingly, the present disclosure also provides a BiTEcomprising one or more amino acid sequences selected from (a) SEQ ID NO:22 and SEQ ID NO: 23, (b) SEQ ID NO: 24 and SEQ ID NO: 25, (c) SEQ IDNO: 26, and (d) SEQ ID NO: 27, or functional variants thereof. In oneembodiment, the BiTE comprises an amino acid sequence having at least50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%,or at least 95% sequence identity to any one of SEQ ID Nos: 22-27.

In a further embodiment, the bispecific antibody binds CD133 and the NKcell surface receptor CD16.

In another embodiment of the present disclosure, the CD133-binding agentis a bispecific antibody comprising antibody variable region RW01 andantibody variable region RW03.

As described above, the CD133-binding agent may have any number ofvalencies and/or specificities. For example, a trispecific and/ortrivalent CD133-binding agent can be prepared (see, e.g. Tutt et al.,1991).

As further described above, embodiments of the CD133-binding agents alsoinclude CD133-binding chimeric antigen receptors (CARs).

Accordingly, provided herein is a chimeric antigen receptor comprising(i) a CD133-binding agent of the disclosure and (ii) a CAR signalingdomain comprising one or more immune cell receptor signaling domains.Chimeric antigen receptors are engineered receptors wherein apolypeptide comprising a CD133-binding variable region of the presentdisclosure, for example a CD133-binding scFv, is fused, for example viaa hinge domain and transmembrane domain, to a CAR signaling domaincomprising one or more intracellular signaling domains of one or moreimmune cell receptors. The CAR can be a monomeric polypeptide (e.g.anti-CD133 scFv-based) or a multimeric polypeptide (e.g. anti-CD133Fab-based). Expression of such a CAR in an immune effector cell allowstargeting of the immune cell to a cell expressing surface CD133, wherebinding of the CAR to the cell surface expressed CD133 activateseffector functions of the immune effector cell.

In one embodiment, the CAR signaling domain comprises a signaling domainof the T cell co-receptor CD3 (e.g. CD3zeta or CD3gamma). In anotherembodiment, the CAR comprises a signaling domain of the T cellco-receptor CD3 fused to a signaling domain of one or more T cellcostimulatory molecules (e.g. CD28, 4-1BB, CD137, OX40, ICOS and/orCD27). In yet another embodiment, the CAR signaling domain comprisesCD3zeta, and portions of CD8 and CD28. In another embodiment, the CARsignaling domain comprises a human CD8 leader sequence and a CD8atransmembrane domain. In one embodiment the CAR comprises a CD28signaling domain, and a terminal CD3zeta signaling domain. In a furtherembodiment, the CAR signaling domain comprises a CD3zeta signalingdomain, a 4-1BB signaling domain, and a CD28 signaling domain. Differentconfigurations of a scFv comprised in the CAR are contemplated includingVL-linker-VH and VH-linker-VL. The construction of suitable CARs andtheir use for targeting antigen expressing cells, commonly referred toin the art as “CAR T cell therapy”, is well known in the art (see, e.g.Maus and June, 2016; Abate-Daga and Davila, 2016; Resetca et al., 2016;and Wang and Riviere, 2016).

In an additional embodiment, the CD133-binding agent is a phage-Fab orphage-scFv, where the Fab or scFv specifically binds CD133. Thedisclosure also provides a T cell expressing a CAR as described herein.

It can be desirable to modify a binding agent disclosed herein withrespect to effector function, so as to enhance its effectiveness inbinding/targeting CD133-expressing cells and/or reducing levels of CD133in CD133-expressing cells. For example, where the binding agentcomprises an antibody Fc region, such as an antibody, cysteineresidue(s) can be introduced into the COOH terminal of the Fc region,thereby allowing interchain disulfide bond formation between antibodymonomers in this region. The homodimeric antibody thus generated canhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC) (see, e.g. Caron et al., 1992; and Shopes, 1992).Alternatively, an antibody can be engineered that has dual Fc regionsand can thereby have enhanced complement lysis and ADCC capabilities(see, e.g. Stevenson et al., 1989). Functional variants of theCD133-binding agents described herein are also encompassed by thepresent disclosure. The term “functional variant” as used hereinincludes modifications or chemical equivalents of the amino acid andnucleic acid sequences disclosed herein that perform substantially thesame function as the polypeptides or nucleic acid molecules disclosedherein in substantially the same way. For example, functional variantsof polypeptides disclosed herein include, without limitation,conservative amino acid substitutions.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residue aresubstitutions that change an amino acid to a different amino acid withsimilar biochemical properties (e.g. charge, hydrophobicity and size).Variants of polypeptides also include additions and deletions to thepolypeptide sequences disclosed herein. In addition, variant nucleotidesequences include analogs and derivatives thereof. A variant of thebinding agents disclosed herein include agents that bind to the sameantigen or epitope as the binding agents.

In one embodiment, the present disclosure includes functional variantsto the amino acid sequences disclosed herein. In particular, thedisclosure provides functional variants of the amino acid sequences ofthe light chain and heavy chain of IgG RW01 (SEQ ID NO: 2 and SEQ ID NO:3, respectively), functional variants of the amino acid sequences of theCDRs of antibody variable region RW01 (SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11), and functionalvariants of the amino acid sequences corresponding to SEQ ID NO: 32 andSEQ ID NO: 33 of the heavy chain of antibody variable region RW01. Thedisclosure further particularly provides functional variants of theamino acid sequences of the light chain and heavy chain of IgG RW03 (SEQID NO: 4 and SEQ ID NO: 5, respectively), functional variants of theamino acid sequences of the CDRs of antibody variable region RW03 (SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16and SEQ ID NO: 17), and functional variants of the amino acid sequencescorresponding to SEQ ID NO: 34 and SEQ ID NO: 35 of the heavy chain ofantibody variable region RW03.

In another embodiment, the present disclosure includes functionalvariants to the nucleic acid sequences that encode the amino acidsequences disclosed herein. Particularly provided are functionalvariants of the nucleotide sequences encoding the light chain and heavychain of IgG RW01 (SEQ ID NO: 18 and SEQ ID NO: 19, respectively),functional variants of the nucleotide sequences encoding the light chainand heavy chain variable domains of antibody variable region RW01 (SEQID NO: 52 and SEQ ID NO: 53, respectively), functional variants of thenucleotide sequences encoding the amino acid sequences of the CDRs ofantibody variable region RW01 (SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41), and functionalvariants of the nucleotide sequences encoding the amino acid sequencescorresponding to SEQ ID NO: 32 and SEQ ID NO: 33 of the heavy chain ofantibody variable region RW01 (SEQ ID NO: 42 and SEQ ID NO: 43,respectively).

The disclosure further particularly provides functional variants of thenucleotide sequences encoding the light chain and heavy chain of IgGRW03 (SEQ ID NO: 20 and SEQ ID NO: 21, respectively), functionalvariants of the nucleotide sequences encoding the light chain and heavychain variable domains of antibody variable region RW03 (SEQ ID NO: 54and SEQ ID NO: 55, respectively), functional variants of the nucleotidesequences encoding the amino acid sequences of the CDRs of antibodyvariable region RW03 (SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49), and functional variants ofthe nucleotide sequences encoding the amino acid sequences correspondingto SEQ ID NO: 34 and SEQ ID NO: 35 of the heavy chain of antibodyvariable region RW03 (SEQ ID NO: 50 and SEQ ID NO: 51, respectively).

In addition, the functional variants include nucleotide sequences thathybridize to the nucleic acids encoding the amino acid sequences of thepresent disclosure, or the complement thereof, under at least moderatelystringent hybridization conditions. Such functional variants includenucleotide sequences that hybridize to SEQ ID NO: 18, SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54 or SEQ ID NO: 55, or the complement thereof,under at least moderately stringent hybridization conditions.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41 (%(G+C)−600/l), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In someembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm−5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

In one embodiment, the variant amino acid sequences of the amino acidsequences disclosed herein comprise sequences having at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90%, or atleast 95% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34 or SEQ ID NO: 35.

In another embodiment, the variant amino acid sequences of the aminoacid sequences disclosed herein comprise sequences having at least 50%,or at least 60%, or at least 70%, or at least 80%, or at least 90%, orat least 95% sequence identity to the framework regions of SEQ ID NOS:2, 3, 4 or 5. In another embodiment, the variant nucleotide sequencesencoding the amino acid sequences disclosed herein comprise sequenceshaving at least 50%, or at least 60%, or at least 70%, or at least 80%,or at least 90%, or at least 95% sequence identity to SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ IDNO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 or SEQ ID NO: 55.

In another embodiment, the variant nucleotide sequences encoding aminoacid sequences comprising heavy and light chain variable domainsdisclosed herein comprise sequences having at least 50%, or at least60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%sequence identity to the nucleotide sequences encoding such amino acidsequences, including SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 21, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two amino acid sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g. gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. One non-limiting example of a mathematical algorithm utilizedfor the comparison of two sequences is the algorithm of Karlin andAltschul, 1990, modified as in Karlin and Altschul, 1993. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990. BLAST nucleotide searches can be performed withthe NBLAST nucleotide program parameters set, e.g. for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the present disclosure. BLAST protein searches can beperformed with the XBLAST program parameters set, e.g. to score-50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997. Alternatively, PSI-BLAST can be used to performan iterated search which detects distant relationships betweenmolecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs,the default parameters of the respective programs (e.g. of XBLAST andNBLAST) can be used (see, e.g. the NCBI website). Another non-limitingexample of a mathematical algorithm utilized for the comparison ofsequences is the algorithm of Myers and Miller, 1988. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

Nucleic Acids and Vectors

Also provided are nucleic acids encoding the antibody variable regionsdescribed herein and nucleic acids encoding polypeptides comprisingthese antibody variable regions. As used herein, the term “nucleicacids” includes isolated nucleic acids.

In particular the present disclosure provides nucleic acids encoding theCDR regions of antibody variable region RW01 as set out in SEQ ID NOs:36-41, and functional variants thereof; and nucleic acids encoding theamino acid sequences of the heavy chain variable domain of RW01 as setout in SEQ ID NOs: 42 and 43, and functional variants thereof. Alsoprovided are nucleic acids encoding the CDR regions of RW03 as set outin SEQ ID NOs: 44-49, and functional variants thereof; and nucleic acidsencoding the amino acid sequences of the heavy chain variable domain ofRW03 as set out in SEQ ID NOs: 50 and 51, and functional variantsthereof.

Further provided is a nucleic acid (SEQ ID NO: 52) encoding the lightchain variable domain of antibody variable region RW01, and functionalvariants thereof. In one embodiment, the nucleic acid encodes (a) theamino acid sequence of SEQ ID NO: 28 or (b) an amino acid sequencehaving at least 70% sequence identity to the framework regions of SEQ IDNO: 28. Also provided is a nucleic acid (SEQ ID NO: 53) encoding theheavy chain of IgG RW01. In one embodiment, the nucleic acid encodes (a)the amino acid sequence of SEQ ID NO: 29 or (b) an amino acid sequencehaving at least 70% sequence identity to the framework regions of SEQ IDNO: 29.

Further provided is a nucleic acid (SEQ ID NO: 54) encoding the lightchain variable domain of antibody variable region RW03, and functionalvariants thereof. In one embodiment, the nucleic acid encodes (a) theamino acid sequence of SEQ ID NO: 30 or an amino acid sequence having atleast 70% sequence identity to the framework regions of SEQ ID NO: 30.Also provided is a nucleic acid (SEQ ID NO: 55) encoding the heavy chainvariable domain of antibody variable region RW03. In one embodiment, thenucleic acid encodes (a) the amino acid sequence of SEQ ID NO: 31 or anamino acid sequence having at least 70% sequence identity to theframework regions of SEQ ID NO: 31.

The disclosure also provides nucleic acids encoding the light chain andheavy chain of IgG RW01 and IgG RW03 as set out in SEQ ID Nos: 18, 19,20 and 21, and functional variants thereof.

The disclosure also provides nucleic acids encoding the variable domainsof the light chain and heavy chain of antibody variable region RW01 andantibody variable region RW03 as set out in SEQ ID Nos: 52, 53, 54 and55, and functional variants thereof.

Polypeptidic binding agents disclosed herein can be expressed by avector containing a nucleic acid encoding the polypeptide of interestusing methods which are well known and routinely practiced in the art.Accordingly, the present disclosure also provides a vector expressingany of the nucleic acids described herein.

The polypeptidic binding agents can be prepared by constructing anucleic acid encoding a polypeptidic binding agent, inserting theconstruct into an expression vector, and then expressing it inappropriate host cells. Vectors useful for expressing the polypeptidicbinding agents disclosed herein are well known in the art. In oneembodiment, the vector includes suitable translation initiation andtermination signals in operable reading phase with a functional promoterand can comprise one or more phenotypic selectable markers and an originof replication to ensure maintenance of the vector and, if desirable, toprovide amplification within the host. In addition to vectors, thenucleic acids of the present disclosure can be delivered to a cell or asubject via any other method known in the art including, but not limitedto, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters,etc.

Monoclonal Polypeptides/Monoclonal Antibodies

As described above, the CD133-binding agent can be a polypeptidecomprising a CD133-binding antibody variable region, such as an antibodyspecifically comprising antibody variable region RW01 or antibodyvariable region RW03. Accordingly, the disclosure further provides amonoclonal polypeptidic CD133-binding agent of the disclosure, such as amonoclonal CD133-binding antibody of the disclosure.

As used herein, a “monoclonal” polypeptidic CD133-binding agent of thedisclosure refers to a population of identical polypeptidicCD133-binding agent molecules. For example, in the case of a monoclonalpolypeptidic CD133-binding agent of the disclosure comprising aCD133-binding antibody variable region, such as a monoclonalCD133-binding antibody of the disclosure, the CDRs are identical in allthe molecules of the population. Various procedures known within the artmay be used for the production of monoclonal polypeptides, such asmonoclonal antibodies of the disclosure (see, for example, Greenfield,2013). Monoclonal antibodies are commonly alternatively referred tousing the abbreviations “mAb” or “MAb”.

Monoclonal antibodies can be made by recombinant DNA methods, such asthose described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonalantibodies and antigen-binding fragments thereof can be readily isolatedand sequenced using conventional procedures (e.g. by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells serve as a preferred source of such DNA. Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells.

Monoclonal antibodies may also be generated, e.g. by immunizing ananimal with CD133, such as, for example, murine, rat or human CD133 oran immunogenic fragment, derivative or variant thereof. Alternatively,the animal is immunized with cells transfected with a vector containinga nucleic acid molecule encoding CD133 that is expressed and associatedwith the surface of the transfected cells. Alternatively, the antibodiesare obtained by screening a library that contains antibody or antigenbinding domain sequences for binding to CD133. This library is prepared,e.g. in bacteriophage as protein or peptide fusions to a bacteriophagecoat protein that is expressed on the surface of assembled phageparticles and the encoding DNA sequences contained within the phageparticles (i.e., “phage displayed library”). Hybridomas resulting frommyeloma/B cell fusions are then screened for reactivity to CD133.

Monoclonal antibodies may be prepared, for example, using hybridomamethods (see, for example, Kohler and Milstein, 1975). In a hybridomamethod, a mouse, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes can be immunized invitro.

Affinity

Non-covalent interactions occur between an immunoglobulin molecule andan antigen for which the immunoglobulin is specific. The strength, oraffinity of immunological binding interactions can be expressed in termsof the dissociation constant (K_(D)) of the interaction, wherein asmaller K_(D) represents a greater affinity. Immunological bindingproperties of specific polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(K_(on)) and the “off rate constant” (K_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation (see, e.g. Malmqvist, 1993). The ratio ofK_(off)/K_(on) enables the cancellation of all parameters not related toaffinity, and is equal to the dissociation constant K_(D) (see, e.g.Davies et al., 1990).

A bivalent CD133-binding agent disclosed herein, such as a CD133-bindingagent comprising two CD133-binding antibody variable regions (e.g. anantibody or F(ab′)₂), is considered to specifically bind CD133 when thedissociation constant (K_(D)) of the binding is ≤1 micromolar. Amonovalent CD133-binding agent disclosed herein (i.e. which has singleCD133-binding site, such as a single CD133-binding antibody variableregion, e.g. a scFv or a Fab) is said to specifically bind CD133 whenthe dissociation constant (K_(D)) of the binding of the CD133-bindingagent in bivalent form is ≤1 micromolar. Methods for joining monovalentbinding agents of the disclosure for generating suitable bivalent formsthereof are well known in the art (e.g. where the monovalent agentcomprises a single antibody variable region, production of bivalentantibodies/F(ab′)₂ comprising two copies of the antibody variableregion; or e.g. using suitable linkers, such as polypeptide linkers,nucleic acid linkers or chemically synthesized linkers).

In various embodiments, the CD133-binding agent binds CD133 with adissociation constant (K_(D)) of ≤1 micromolar, ≤900 nM, ≤800 nM, ≤700nM, ≤600 nM, ≤500 nM, 400 nM, ≤300 nM, ≤200 nM, ≤100 nM, ≤90 nM, ≤80 nM,≤70 nM, ≤60 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤2 nM, ≤1 nM, ≤50.9 nM, ≤0.8 nM, ≤0.7 nM, ≤0.6nM, 0.5 nM, ≤0.4 nM to 0.3 nM, ≤0.2 nM, or ≤100 pM to about 1 pM.

In further various embodiments, the CD133-binding agent binds CD133 witha dissociation constant (K_(D)) of ≤1 micromolar to 100 nM, ≤100 nM to10 nM, ≤10 nM to 1 nM, ≤1 nM to 0.1 nM, or ≤0.1 nM to 10 pM.

In additional various embodiments, the CD133-binding agent binds CD133with a dissociation constant (K_(D)) of ≤3 nM to 2 nM, ≤2.6 nM to 2.4nM, ≤2.5 nM, about 2.5 nM, 2 nM to 1 nM, ≤0.6 nM to 0.4 nM, ≤0.5 nM, orabout 0.5 nM.

As disclosed herein, the dissociation constant KD for the binding of abivalent CD133-binding agent, such as a CD133-binding antibody of thedisclosure or a monovalent CD133-binding agent in bivalent form, isconsidered to approximately correspond to the half-maximal concentration(“EC50”) of the CD133-binding agent required to saturate binding to apopulation of CD133-overexpressing cells, such as HEK293-CD133 cells, asdetermined via flow cytometry (see Example 3, below). This method isuseful for determining the affinity of a binding agent for a cellsurface molecule which cannot be suitably purified, as is often the casewith transmembrane proteins, such as CD133. Alternate methods ofdetermining a dissociation constant (KD) for binding of a CD133-bindingagent to cell surface-expressed CD133 include radioligand bindingassays, and similar assays known to those skilled in the art.Alternately, where CD133 or a portion thereof bound by a CD133-bindingagent of the disclosure is available in purified form, the dissociationconstant (KD) can be measured by assays such as surface plasmonresonance (SPR; Biacore™) assay and other suitable assays known in theart.

As described above, the disclosure provides a CD133-binding agent whichspecifically binds a CD133 epitope bound by antibody IgG RW01 and/or aCD133 epitope bound by antibody IgG RW03.

Any one of various methods known in the art can be used to identify aCD133-binding agent which specifically binds a CD133 epitope bound byantibody IgG RW01 and/or a CD133 epitope bound by antibody IgG RW03. Aperson skilled in the art will appreciate that binding assays such as acompetition binding assay can be used for this purpose. Those skilled inthe art will recognize that it is possible to determine, without undueexperimentation, if a binding agent specifically binds a CD133 epitopebound by antibody IgG RW01 and/or a CD133 epitope bound by antibody IgGRW03 by ascertaining whether the binding agent prevents antibody IgGRW01 and/or antibody IgG RW03 from binding to human CD133. If thebinding agent being tested competes with antibody IgG RW01 and/orantibody IgG RW03, as shown by a decrease in binding to human CD133 byantibody IgG RW01 and/or antibody IgG RW03, then the binding agent bindsto the same epitope as antibody IgG RW01 and/or antibody IgG RW03.Methods for the testing the specificity of binding agents include, butare not limited to, enzyme linked immunosorbent assay (ELISA) and otherimmunologically mediated techniques known within the art.

In one embodiment, the CD133-binding agent which specifically binds aCD133 epitope bound by antibody IgG RW01 and/or a CD133 epitope bound byantibody IgG RW03 is a CD133-binding agent which competes with antibodyIgG RW01 and/or antibody IgG RW03 for binding to the surface ofCD133-expressing cells. In one embodiment, the CD133 epitope is a humanCD133 epitope. For example, a CD133-binding agent which specificallybinds a CD133 epitope bound by antibody IgG RW01 and/or a CD133 epitopebound by antibody IgG RW03 will partially or fully inhibit binding ofantibody IgG RW01 and/or antibody IgG RW03 to CD133-expressing cellspre-incubated with a saturating concentration of such a binding agent,as determined via flow cytometry analogously to the experimentsdisclosed in Example 7. Alternately, a CD133-binding agent whichspecifically binds a CD133 epitope bound by antibody IgG RW01 and/or aCD133 epitope bound by antibody IgG RW03 will partially or fully inhibitbinding of antibody IgG RW01 and/or antibody IgG RW03 to denaturedCD133, e.g. denatured CD133 electroblotted onto a membrane (e.g.nitrocellulose or PVDF) following electrophoretic separation underreducing conditions of denatured protein of whole cell lysate (see, e.g.Example 6), where the membrane has been pre-incubated with a saturatingconcentration of such binding agent.

In another embodiment, the CD133-binding agent which specifically bindsa CD133 epitope bound by antibody IgG RW01 and/or a CD133 epitope boundby antibody IgG RW03 is a monovalent CD133-binding agent (e.g. a Fab ora scFv) which competes with Fab comprising antibody variable region RW01and/or Fab comprising antibody variable region RW03 for binding to thesurface of CD133-expressing cells. In one embodiment, the CD133 epitopeis a human CD133 epitope. For example, a monovalent CD133-binding agentwhich specifically binds a CD133 epitope bound by antibody IgG RW01and/or a CD133 epitope bound by antibody IgG RW03 will partially orfully inhibit binding of Fab comprising antibody variable region RW01and/or Fab comprising antibody variable region RW03 to CD133-expressingcells pre-incubated with a saturating concentration of such a bindingagent, as determined via flow cytometry analogously to the experimentsdisclosed in Example 7. Alternately, the monovalent agent will partiallyor fully inhibit binding of Fab comprising antibody variable region RW01and/or Fab comprising antibody variable region RW03 to denatured CD133,e.g. denatured CD133 electroblotted onto a membrane (e.g. nitrocelluloseor PVDF) following electrophoretic separation under reducing conditionsof denatured protein of whole cell lysate (see, e.g. Example 6), wherethe membrane has been pre-incubated with a saturating concentration ofsuch binding agent following electroblotting. In one embodiment, theCD133 is human CD133.

Detection Agents

The binding agents described herein are optionally labeled with adetection agent. As used herein, the term “detection agent” refers toany agent that allows the presence of the binding agent to be detectedand/or quantified. Examples of detection agents include, but are notlimited to, peptide tags, enzymes (for example, HRP or alkalinephosphatase), proteins (for example phycoerythrin orbiotin/streptavidin), magnetic particles, chromophores, fluorescentmolecules, chemiluminescent molecules, radioactive labels and dyes. Thebinding agent may be labeled directly or indirectly with the detectionagent.

Humanized Antibodies

The nucleotide sequence encoding a non-human, e.g. murine, CD133-bindingagent disclosed herein can be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous non-human, e.g. murine, sequences (see, e.g.U.S. Pat. No. 4,816,567; and Morrison, 1994) or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodydisclosed herein, or can be substituted for the variable domains of oneantigen-combining site of an antibody disclosed herein to create achimeric bivalent antibody.

The non-human binding agents comprising Fc regions, e.g. non-humanantibodies, described herein may be humanized in order to make thembetter tolerated for use in humans. For example, amino acid residues inthe framework regions may be humanized by replacing them with amino acidresidues and the human framework regions as long as the replacement doesnot impair the ability of the binding agents to bind to CD133 (see, e.g.Vincke et al., 2008).

It will be appreciated that murine antibodies or antibodies from otherspecies can be humanized or primatized using techniques well known inthe art (see, e.g. Winter and Harris, 1993; and Wright et al., 1992). Anantibody may be engineered by recombinant DNA techniques to substitutethe CH1, CH2, CH3, hinge domains, and/or the framework domain with thecorresponding human sequence (see, e.g. WO 92102190 and U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,792; 5,714,350; and 5,777,085).Also, the use of Ig cDNA for construction of chimeric immunoglobulingenes is known in the art (see, e.g. Liu et al., 1987a; and Liu et al.,1987b). mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. Human C region genes are readily available from known clones.The choice of isotype will be guided by the desired effecter functions,such as complement fixation, or activity in antibody-dependent cellularcytotoxicity. Either of the human light chain constant regions, kappa orlambda, may be used. The chimeric, humanized antibody may then beexpressed by conventional methods.

As described above, the CD133-binding agent may be a human antibody.Fully human antibodies are antibody molecules in which the entiresequence of both the light chain and the heavy chain, including theCDRs, arise from human genes. Such antibodies are termed “humanantibodies” or “fully human antibodies” herein. Human monoclonalantibodies can be prepared by using the trioma technique; the humanB-cell hybridoma technique (see, e.g. Kozbor, et al., 1983), and the EBVhybridoma technique to produce human monoclonal antibodies (see, e.g.Cole et al., 1985). Human monoclonal antibodies may be utilized and maybe produced by using human hybridomas (see, e.g. Cote, et al., 1983) orby transforming human B-cells with Epstein Barr Virus in vitro (see,e.g. Cole et al., 1985).

A CD133-binding polypeptide comprising a CD133-binding antibody variableregion, such as a CD133-binding scFv or CD133-binding Fab may bedeveloped, for example, using phage-display methods using antibodiescontaining only human sequences. Such approaches are well-known in theart (see, e.g. WO92/01047 and U.S. Pat. No. 6,521,404, which are herebyincorporated by reference). In this approach, a combinatorial library ofphage carrying random pairs of light and heavy chains are screened usingnatural or recombinant source of CD133 or fragments thereof. In anotherapproach, an antibody or fragment can be produced by a process whereinat least one step of the process includes immunizing a transgenic,non-human animal with a CD133 protein. In this approach, some of theendogenous heavy and/or kappa light chain loci of thisxenogeneic/non-human animal have been disabled and are incapable of therearrangement required to generate genes encoding immunoglobulins inresponse to an antigen. In addition, at least one human heavy chainlocus and at least one human light chain locus have been stablytransfected into the animal. Thus, in response to an administeredantigen, the human loci rearrange to provide genes encoding humanvariable regions immunospecific for the antigen. Upon immunization,therefore, the animal produces B-cells that secrete fully humanimmunoglobulins.

A variety of techniques are well-known in the art for producingxenogeneic/non-human animals (see, e.g. U.S. Pat. Nos. 6,075,181 and6,150,584, which are hereby incorporated by reference; Green et al.,1994, which is hereby incorporated by reference in its entirety; U.S.Pat. Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598;Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2;European Patent No., EP 0 463 151 B1; and International PatentApplication Nos. WO 94/02602, WO 96/34096, WO 98/24893, and WO00/76310).

Alternatively, a “minilocus” approach may be used, in which an exogenousIg locus is mimicked through the inclusion of pieces (individual genes)from the Ig locus (see, e.g. U.S. Pat. Nos. 5,545,806, 5,545,807,5,591,669, 5,612,205, 5,625,825, 5,625,126, 5,633,425, 5,643,763,5,661,016, 5,721,367, 5,770,429, 5,789,215, 5,789,650, 5,814,318,5,877,397, 5,874,299, 6,023,010, and 6,255,458; European Patent No. 0546 073 B1; and International Patent Application Nos. WO 92/03918, WO92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO94/25585, WO 96/14436, WO 97/13852, and WO 98/24884). Thus, one or moreVH genes, one or more DH genes, one or more JH genes, a mu constantregion, and a second constant region (preferably a gamma constantregion) are formed into a construct for insertion into an animal.

Generation of human antibodies from mice in which, through microcellfusion, large pieces of chromosomes, or entire chromosomes, have beenintroduced, has also been demonstrated (see, e.g. European PatentApplication Nos. 773 288 and 843 961).

Immunoconjugates

The present disclosure also includes an immunoconjugate comprising (1) aCD133-binding agent, optionally an antibody or an antibody antigenbinding fragment, that has been attached to (2) an effector agent. Asused herein, the term “immunoconjugate” encompasses CD133-binding agentsof the disclosure which do not comprise an antibody variable region, andfurther encompasses CD133-binding agents disclosed herein which comprisean antibody variable region.

In one embodiment, the effector agent is a label, which can generate adetectable signal, directly or indirect. Examples of labels includeradioactive isotopes (i.e., a radioconjugate).

In another embodiment, the effector agent is a therapeutic agent.Therapeutic agents include, but are not limited to, cancer therapeuticagents/antineoplastic agents. In yet another embodiment, the therapeuticagent is a toxin.

The term “cancer therapeutic agent” or “antineoplastic agent” is usedherein to refer to agents that have the functional property ofdecreasing levels of CD133 in cancer cells, such as pancreatic cancercells, colorectal cancer cells, breast cancer cells, colon cancer cells,gastric cancer cells, prostate cancer cells, liver cancer cells, lungcancer cells, melanoma cells, brain cancer cells (optionallyglioblastoma or medulloblastoma cells) and head and neck squamous cellcarcinoma cells.

The toxin may be an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or a fragment thereof. Toxins and fragmentsthereof that can be used include diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

Radioconjugated CD133-binding agents of the disclosure, such asantibodies of the disclosure, may be employed to bind radionuclides toCD133-expressing cells, for example to visualize the cells or as acytotoxic treatment of the cells. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude 212Bi, 131I, 131In, 90Y, and 186Re.

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the polypeptidic CD133-bindingagents of the disclosure, such as those comprising an antibody variableregion (e.g. antibodies or antibody fragments comprising a CD133-bindingantibody variable region) (see, for example, Cruse and Lewis, 1989, theentire contents of which are incorporated herein by reference). Couplingmay be accomplished by any chemical reaction that will bind a moiety anda CD133-binding agent of the disclosure, so long as these retain theirrespective activities/characteristics for the intended use thereof. Thislinkage can include many chemical mechanisms, for instance covalentbinding, affinity binding, intercalation, coordinate binding andcomplexation.

For example, conjugates of a polypeptidic CD133-binding agent of thedisclosure, such as an antibody and an effector agent can be made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody (see, e.g. WO94/11026).

Pharmaceutical Compositions

The disclosure also provides pharmaceutical compositions comprising aCD133-binding agent or immunoconjugate or radioconjugate describedherein as an active ingredient and a pharmaceutically acceptablecarrier.

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Suitable carriers are described in themost recent edition of Remington's Pharmaceutical Sciences, a standardreference text in the field, which is incorporated herein by reference.Optional examples of such carriers or diluents include, but are notlimited to, water, saline, ringer's solutions, dextrose solution, and 5%human serum albumin.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g. intravenous, intradermal, subcutaneous, oral(e.g. inhalation), transdermal (i.e., topical), transmucosal, and rectaladministration.

In one embodiment, the active ingredient is prepared with a carrier thatwill protect it against rapid elimination from the body, such as asustained/controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

In one embodiment, oral or parenteral compositions are formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active ingredient calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms aredictated by and directly dependent on the unique characteristics of theactive ingredient and the particular therapeutic effect to be achieved,and the limitations inherent in the art of preparing such an activeingredient for the treatment of individuals.

The formulation can also contain more than one active ingredient asnecessary for the particular indication being treated, optionally thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the pharmaceutical composition cancomprise an agent that enhances its function, such as, for example, acytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitoryagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

Methods and Uses:

The disclosure also provides uses and methods relating to theCD133-binding agents described herein.

Detecting CD133-Expressing Cells

The CD133-binding agents, immunoconjugates and pharmaceuticalcompositions of the present disclosure are useful for detecting cellsthat express CD133. Accordingly, the disclosure provides a use of theCD133-binding agents described herein for targeting, binding and/ordetecting CD133-expressing cells. Optionally, the cells are cancercells, including, but not limited to, pancreatic cancer cells,colorectal cancer cells, breast cancer cells, colon cancer cells,gastric cancer cells, prostate cancer cells, liver cancer cells, lungcancer cells, melanoma cells, brain cancer cells and head and necksquamous cell carcinoma cells.

In one embodiment, the CD133-binding agents, immunoconjugates, andpharmaceutical compositions described herein are useful for targeting,binding and/or detecting cell surface expression of CD133-expressingcells.

In another embodiment, the CD133-binding agents, immunoconjugates andpharmaceutical compositions described herein are useful for targeting,binding, detecting and/or localizing intracellular CD133.

In another embodiment, the CD133-binding agents, immunoconjugates andpharmaceutical compositions described herein are useful for targeting,binding and/or detecting CD133 in cell lysates.

In yet another embodiment, the CD133-binding agents, immunoconjugatesand pharmaceutical compositions described herein are useful fordetecting and/or quantitating levels of expression of CD133 in a sample,optionally in a CD133 expressing cell. In one embodiment, theCD133-binding agents, immunoconjugates and pharmaceutical compositionsare used to detect and/or quantitate cellular CD133 levels. In anotherembodiment, the CD133-binding agents, immunoconjugates andpharmaceutical compositions are useful for detecting and/or quantitatingcell surface CD133 levels.

The CD133-binding agents of the disclosure may be used fordetecting/quantitating both native/cell-surface expressed as well asdenatured CD133. Overexpression of CD133 often correlates with acancerous phenotype. For example, Western blotting detection of CD133protein levels in denatured whole cell lysates using CD133-bindingagents of the disclosure can be used to characterize/confirm the abilityof a treatment to reduce the metastatic capacity of CD133-expressingcancer cells, since reduced total cellular CD133 protein levels has beenshown to correlate with reduced metastatic capacity of the cells (see,e.g. Rappa et al. 2008).

In general, the use of binding agents for detection of analytes, such asintracellular, total cellular or surface-expressed CD133 protein, iswell known in the art and may be achieved through the application ofnumerous approaches. These methods are generally based upon thedetection of a label or marker, such as radioactive, fluorescent,biological and enzymatic tags. Examples of methods include, but are notlimited to, Western blotting, enzyme linked immunosorbent assay (ELISA),immunofluorescence, immunohistochemistry and flow cytometry.

The CD133-binding agents, immunoconjugates and pharmaceuticalcompositions of the present disclosure are also useful for reducingand/or eliminating the level or amount of CD133 protein in a cell.Optionally, the cell is a CD133-positive cancer cell, including, but notlimited to, a pancreatic cancer cell, colorectal cancer cell, breastcancer cell, colon cancer cell, gastric cancer cell, prostate cancercell, liver cancer cell, pancreatic cancer cell, lung cancer cell,melanoma cell, brain cancer cell (optionally a glioblastoma ormedulloblastoma cell) and head and neck squamous cell carcinoma cell.Reduction of total cellular CD133 protein levels in cancer cells can beused to reduce the metastatic capacity thereof (see, e.g. Rappa et al.2008). The CD133 protein of which the levels are reduced is optionallycell surface-expressed and/or intracellular CD133. The CD133 protein ina CD133-expressing cell is optionally reduced by at least 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98or 99%; or by 100%.

Targeting CD133-Expressing Cells to Immune Cells

Further, the CD133-binding agents, immunoconjugates and pharmaceuticalcompositions of the present disclosure are useful for engaging,targeting and/or binding cells of the immune system.

For example, in one embodiment described above, the CD133-binding agentis a bispecific antibody where one of the binding specificities is forCD133 and the other binding specificity is for an antigen expressed onan immune cell such as a T cell, macrophage or NK cell. As describedabove, one example of a bispecific antibody that targets T cells is abispecific T-cell engager (BiTE).

In another embodiment described above, the CD133-binding agent is aCD133-binding chimeric antigen receptor (CAR) which includes aCD133-binding agent of the disclosure, such as a CD133-binding scFv asits antigen-binding/targeting domain.

The construction of suitable CARs and their use for targeting antigenexpressing cells, commonly referred to in the art as “CAR T celltherapy”, is well known in the art (see, e.g. Maus and June, 2016;Abate-Daga and Davila, 2016; Resetca et al., 2016; and Wang and Riviere,2016). Accordingly, the bispecific antibodies and chimeric antigenreceptors described herein are useful for targeting immune effectorcells to CD133-expressing cells (CD133+ cells).

Also provided are methods for targeting CD133+ cells comprising exposingthe CD133+ cells to an immune effector cell expressing a CAR of thedisclosure, or to a combination of a bispecific antibody of thedisclosure and an immune effector cell specifically bound by thebispecific antibody.

Targeting immune effector cells to CD133+ cells through these methodsmay be useful for eliminating, and/or shifting the phenotype of, CD133+cancer cells from a cancerous phenotype towards a less cancerous ornon-cancerous phenotype. In addition, targeting immune effector cells toCD133+ cells may be useful for treating diseases where CD133 isexpressed or overexpressed such as cancer.

Diagnostic Methods

The CD133-binding agents disclosed herein are useful in thedetection/quantitation of CD133 in patient samples or in control samplesof healthy individuals and accordingly may be useful diagnostics. Forexample, the binding agents of the disclosure can be used todetect/quantitate total cellular expression of CD133 and/or cell-surfaceexpressed CD133. As used herein, the term “diagnostics” encompassesscreening, stratification, monitoring and the like.

In one embodiment, the CD133-binding agents are used to detect CD133expressing cells, optionally cancer cells such as pancreatic cancercells, colorectal cancer cells, breast cancer cells, colon cancer cells,gastric cancer cells, prostate cancer cells, liver cancer cells, lungcancer cells, melanoma cells, brain cancer cells and head and necksquamous cell carcinoma cells.

In another embodiment, the CD133-binding agents are used fordetecting/quantitating cell surface expression of CD133. In anotherembodiment, the CD133-binding agents are used for detecting/quantitatingintracellular expression of CD133. In another embodiment, theCD133-binding agents described herein can be used to detect/quantitateexpression of CD133 in a sample.

For example, CD133-binding agents of the disclosure, such as theantibodies and antibody fragments of the disclosure, may be used forpracticing any one of various assays, e.g. immunofluorescence, flowcytometry or ELISAs, to detect/quantitate CD133 levels in a sample.

In one embodiment, the sample is a patient sample, such as a cancersample from a cancer patient. Alternately, the sample may be a controlsample from a healthy individual. Embodiments of the sample include butare not limited to, a sample of cultured cells, cultured cellsupernatant, cell lysate, serum, blood plasma, biological fluid orbiological tissue. In other embodiments, the sample is obtained from acancer. In certain embodiments, the sample is a biopsy sample.

Treatment of Cancer

CD133 has been shown to play an important role in various cancers. Forexample, CD133 has been identified as a marker for cancer stem cells(CSCs) in brain tumors (Singh et al., 2003) and it has been demonstratedthat as few as 100 CD133+ cells from brain tumor fractions aresufficient to generate a tumor in NOD/SCID mice (Singh et al., 2004).Additionally, CD133+ glioma cells have been shown to have increasedresistance to radiation in a DNA checkpoint dependent manner as comparedwith CD133-negative (CD133-) cells (Bao et al., 2006). Pancreatic CSCshave been isolated using anti-CD133 antibodies and it has beendemonstrated that these cells are tumorigenic and highly resistant tostandard chemotherapy (Hermann et al., 2007). Similarly, it has alsobeen shown that increased CD133 expression in pancreatic cancer cellscorrelates with a more aggressive nature including increased migrationand invasion and heightened tumor aggressiveness (Moriyama et al.,2010). Stem-like cells from prostate cancer tissues have been identified(Collins, 2005) and it has been demonstrated that CD133+ cells isolatedfrom prostate cancer tissues and in an immortalized prostate cancer cellline exhibit stem cell features (Miki et al., 2007; Wei et al., 2007),however other studies have failed to confirm this (Missol-Kolka et al.,2010) and have not reported the stem-cell characteristics that othersattribute to pancreatic cancer lines like the DU145 line (Pfeiffer &Schalken, 2010). Another cancer type for which CD133 is used as a CSCmarker is colorectal carcinoma. Separate groups have identified CD133 asa marker of CSCs in colon cancer (O'Brien et al., 2006; Ricci-Vitiani etal., 2006). It has been demonstrated that CD133+ cells readilyrecapitulate tumors in SCID mice (Ricci-Vitiani et al., 2006), and thatthere is an enrichment of colon cancer initiating cells purified CD133+cells compared to unfractionated tumor cells (O'Brien et al., 2006). Ithas been demonstrated that both CD133+ and CD133-negative cells fromcolon metastases are able to form colon-spheres and recapitulate tumorsin NOD/SCID mice (Shmelkov, 2004). Additionally, several groups haveconcluded that CD133 is associated with worse clinical prognosis,disease progression and metastasis and that CD133 protein can be used asan independent prognostic marker for colorectal cancer patients (Horstet al., 2009). However, other groups have failed to find a relationshipbetween CD133 and disease progression or survival in colon cancerpatients but have instead found a relationship between the expression ofthe protein and tumor stage (Lugli et al., 2010).

It has been demonstrated that downregulation of CD133 in the metastaticmelanoma cell line FEMX-I resulted in slower cell growth, decreased cellmotility, decreased ability to form spheres in stem-cell growthconditions and a reduced metastatic capacity of tumor xenografts (Rappaet al., 2008). It has been shown that the anti-CD133 antibody AC133conjugated to monomethyl auristatin F inhibits the growth ofhepatocellular and gastric cancer cells in vitro (Smith et al., 2008),it was further demonstrated that secondary antibody conjugated tosaporin in the presence of AC133 is toxic to FEMX-I cells but notcontrol human fibroblasts (Rappa et al., 2008). Additionally, AC133directly conjugated to saporin has been shown to be more effectiveagainst FEMX-I cells than FEMX-I cells with CD133 expression knockeddown (Rappa et al., 2008). It was observed that in cells in which CD133is knocked down, genes that became upregulated coded for wnt inhibitors(Mak et al., 2012b).

In addition, numerous studies have implicated CD133+ brain tumorinitiating cells (BTICs) as drivers of chemo- and radio-resistance inglioblastoma (GBM). It has also recently been demonstrated that aCD133-driven gene signature is predictive of poor overall survival(Venugopal et al, 2015) and targeting CD133+ treatment refractory cellsmay be an effective strategy to block GBM recurrence.

Accordingly, these results provide support for targeting CD133 as aneffective therapeutic strategy and as an effective diagnostics strategy.In addition, the present inventors have described a CAR T-cell-basedstrategy whereby CD133+ GBM cells are specifically targeted and killed.The present inventors have also shown that a BiTE antibody thatredirects human polyclonal T cells to CD133+ GBM cells induces a potentanti-tumor response.

Accordingly, the CD133-binding agents and pharmaceutical compositions ofthe present disclosure are useful for treating or preventing a cancer,for example a metastatic melanoma, brain, prostate, pancreatic and/orcolon/colorectal cancer. In one embodiment, the cancer is aglioblastoma. In another embodiment, the cancer is a medulloblastoma.

In another embodiment, the cancer is a CD133-positive cancer (alsoreferred to as a CD133-expressing cancer). In one embodiment, aCD133-positive cancer is defined as a cancer with greater than 80%, 85%,90%, 95% or 99% CD133-positive cells (i.e., CD133-expressing cells). Thepercentage of cells expressing CD133 may be determined, for example, ina tumor cell culture. Accordingly, in particular embodiments, the canceris a CD133-positive glioblastoma or a CD133-positive medulloblastoma. Inanother embodiment, the cancer is a glioblastoma detectably expressingCD133 or a medulloblastoma detectably expressing CD133.

In one embodiment, the CD133-binding agents and pharmaceuticalcompositions described herein are used in a method for treating orpreventing cancer, the method comprising administering an effectiveamount of a CD133-binding agent or pharmaceutical composition disclosedherein to an animal or cell in need thereof, optionally wherein thecancer is metastatic melanoma, brain, prostate, pancreatic and/or coloncancer. In one embodiment, the cancer is a glioblastoma or amedulloblastoma.

In another embodiment, an effective amount of a CD133-binding agent orpharmaceutical composition disclosed herein is used for treating orpreventing a cancer, optionally wherein the cancer is metastaticmelanoma, brain, prostate, pancreatic and/or colon cancer. In anotherembodiment, a CD133-binding agent or pharmaceutical compositiondisclosed herein is used in the preparation of a medicament for treatingor preventing a cancer, optionally wherein the cancer is metastaticmelanoma, brain, prostate, pancreatic and/or colon cancer. In oneembodiment, the cancer is a glioblastoma or a medulloblastoma.

In yet another embodiment, an effective amount of a CD133-binding agentor pharmaceutical composition disclosed herein is used for in treatingor preventing a cancer, optionally wherein the cancer is metastaticmelanoma, brain, prostate, pancreatic and/or colon cancer. In oneembodiment, the cancer is a glioblastoma or a medulloblastoma.

As described above, the present disclosure provides immunoconjugatescomprising (1) a CD133-binding agent and (2) an effector agent, wherethe effector agent is optionally a toxin or an anti-neoplastic agent.

Accordingly, the present disclosure provides a method of using animmunoconjugate disclosed herein for treating or preventing a cancer,the method comprising administering an effective amount of animmunoconjugate disclosed herein to an animal or cell in need thereof,optionally wherein the cancer is metastatic melanoma, brain, prostate,pancreatic and/or colon cancer. In one embodiment, the cancer is aglioblastoma or a medulloblastoma.

In one embodiment, an effective amount of an immunoconjugate disclosedherein is used for treating or preventing a cancer, optionally whereinthe cancer is metastatic melanoma, brain, prostate, pancreatic and/orcolon cancer. In another embodiment, an immunoconjugate disclosed hereinis used in the preparation of a medicament for treating or preventing acancer, optionally wherein the cancer is metastatic melanoma, brain,prostate, pancreatic and/or colon cancer. In one embodiment, the canceris a glioblastoma or a medulloblastoma.

The present disclosure also provides CARs that target CD133 expressingcells, and T-cells expressing the CARs. Accordingly, in anotherembodiment, the present disclosure provides a method of using a T cellexpressing a CAR disclosed herein for treating or preventing a cancer,the method comprising administering an effective amount of a T cellexpressing a CAR disclosed herein to an animal or cell in need thereof,optionally wherein the cancer is metastatic melanoma, brain, prostate,pancreatic and/or colon cancer. In one embodiment, the cancer is aglioblastoma or a medulloblastoma.

In one embodiment, an effective amount of a T cell expressing a CARdisclosed herein is used for treating or preventing a cancer, optionallywherein the cancer is metastatic melanoma, brain, prostate, pancreaticand/or colon cancer. In another embodiment, a T cell expressing a CARdisclosed herein disclosed herein is used in the preparation of amedicament for treating or preventing a cancer, optionally wherein thecancer is metastatic melanoma, brain, prostate, pancreatic and/or coloncancer. In one embodiment, the cancer is a glioblastoma or amedulloblastoma.

As used herein, the terms “subject” and “animal” include all members ofthe animal kingdom, in one embodiment the subject is a mammal. In afurther embodiment the subject is a human being. In one embodiment, thesubject is a patient having a disease, such as a cancer, associated withCD133-expressing cells.

The term “a cell” includes a single cell as well as a plurality orpopulation of cells.

An effective amount of a CD133-binding agent, immunoconjugate orpharmaceutical composition of the disclosure relates generally to theamount needed to achieve a therapeutic objective. As noted above, thismay be a binding interaction between the CD133-binding agent and CD133that, in certain cases, interferes with the functioning of CD133.

The amount required to be administered will furthermore depend on thebinding affinity of the CD133-binding agent for CD133, and will alsodepend on the rate at which an administered CD133-binding agent isdepleted from the free volume of the subject to which it isadministered. Common ranges for therapeutically effective dosing of aCD133-binding agent, immunoconjugate or pharmaceutical composition ofthe disclosure may be, by way of non-limiting example, from about 0.1 mgkg body weight to about 50 mg/kg body weight. Common dosing frequenciesmay range, for example, from twice daily to once a week.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular cancer. Alleviation ofone or more symptoms of the cancer indicates that the antibody confers aclinical benefit.

As used herein, “treating a cancer” includes, but is not limited to,reversing, alleviating or inhibiting the progression of the cancer orsymptoms or conditions associated with the cancer. “Preventing a cancer”includes preventing incidence or recurrence of the cancer. “Treating acancer” also includes extending survival in a subject. Survival isoptionally extended by at least 1, 2, 3, 6 or 12 months, or at least 2,3, 4, 5 or 10 years over the survival that would be expected withouttreatment with a CD133-binding agent, immunoconjugate or pharmaceuticalcomposition as described herein. “Treating a cancer” also includesreducing tumor mass and/or reducing tumor burden (for example, braintumor mass and/or brain tumor burden). Optionally, tumor mass and/ortumor burden is reduced by at least 5, 10, 25, 50, 75 or 100% followingtreatment with a CD133-binding agent, immunoconjugate or pharmaceuticalcomposition as described herein. In other embodiments, “treating acancer” includes reducing the aggressiveness, grade and/or invasivenessof a tumor. The tumor is optionally a newly formed tumor or a tumoralready present at the time of treatment.

In one embodiment, the active ingredient may be used in combination withat least one additional therapeutic agent. Accordingly, the applicationprovides a method of preventing or treating a cancer using theCD133-binding agents, immunoconjugates or pharmaceutical compositionsdisclosed herein in combination with at least one additional therapeuticagent. An additional therapeutic agent may be administered prior to,overlapping with, concurrently, and/or after administration of theactive ingredients. When administered concurrently, the CD133-bindingagents, immunoconjugates or pharmaceutical compositions and anadditional therapeutic agent may be administered in a single formulationor in separate formulations, and if administered separately, thenoptionally, by different modes of administration. The combination of oneor more CD133-binding agents, immunoconjugates or pharmaceuticalcompositions and one or more other therapeutic agents maysynergistically act to combat the cancer.

Embodiments of the additional therapeutic agent include additionalCD133-binding agents, additional CD133-binding immunoconjugates,additional CD133-binding pharmaceutical compositions, cytokines, growthfactor inhibitors, immunosuppressants, anti-inflammatory agents,metabolic inhibitors, enzyme inhibitors, anti-neoplastic agents,cytotoxic agents and/or cytostatic agents. Such combination therapiesmay advantageously utilize lower dosages of an administered activeingredient, thus avoiding possible toxicities or complicationsassociated with monotherapy.

Screening Assays

The disclosure also provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., test agents (e.g.peptides, peptidomimetics, small molecules or other drugs) that modulateor otherwise interfere with the binding of a protein disclosed hereinwith the CD133.

The test agents can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection (see, e.g. Lam, 1997).

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of thedisclosure. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1: Cell Selections and Sequencing

In order to attempt to discover novel antibodies capable of specificallybinding CD133, two phage-display libraries, Library F and Library G,were screened for cell surface CD133 binders using the “Cellectseq”method (the Cellectseq method, Library F and Library G have beenpreviously described, e.g. in U.S. patent application Ser. No.13/629,520). Briefly, Library F is a Fab library with diversity in lightchain complementarity-determining region (CDR) 3 and in all three heavychain CDRs, and Library G is scFv library with diversity in all sixCDRs. The cells used for the selections were HEK293 cells engineered tooverexpress CD133 (GenBank™ Accession 043490) for the positive selectionand the parental HEK293 cells were used for the negative selection.After four rounds of positive and negative selection, serial dilutionswere made of the round four output phage (10E-1 to 10E-3) for eachlibrary and used to infect XLI-blue cells. The infected cells wereplated for isolating single colonies to be used in a clonal cell-basedELISA for isolating CD133-specific binders. Clones that bound to theHEK293-CD133 cells and generated an ELISA signal that was at least 1.5fold higher than background binding to HEK293 cells were classified asCD133-specific binders. Sequencing was performed by amplifying theantibody variable (VL and VH) domains from clone DNA with M13-taggedsequencing primers, and the amplified sequences were sequenced viaIllumina sequencing. The full-length amino acid sequence of human CD133expressed in HEK293-CD133 cells is indicated in Table 1. The amino acidsequence coordinates of the three extracellular domains of CD133 are asfollows: Gly20-Glyl08, Ala179-Tyr433 and Gly508-Asn792. The amino acidsequence coordinates of the segment spanning all three extracellulardomains is Gly20-Asn792. The signal sequence corresponding to Met1-Ser19of the full-length sequence is absent from the mature, cell-surfaceexpressed CD133 protein.

TABLE 1 Amino acid sequence of human CD133 (GenBank™ Accession O43490).MALVLGSLLLLGLCGNSFS GGQPSSTDAPKAWNYELPATNYETQDSHKAGPIGILFELVHIFLYVVQPRDFPEDTLRKFLQKAYESKIDYDKPETVILGLKIVYYEAGIILCCVLGLLFIILMPLVGYFFCMCRCCNKCGGEMHQRQKENGPFLRKCFAISLLVICIIISIGIFYGFVANHQVRTRIKRSRKLADSNFKDLRILLNETPEQIKYILAQYNTTKDKAFTDLNSINSVLGGGILDRLRPNIIPVLDEIKSMATAIKETKEALENMNSTLKSLHQQSTQLSSSLTSVKISLRSSLNDPLCLVHPSSETCNSIRLSLSQLNSNPELRQLPPVDAELDNVNNVLRTDLDGLVQQGYQSLNDIPDRVQRQTTTVVAGIKRVLNSIGSDIDNVTQRLPIQDILSAFSVYVNNTESYIHRNLPTLEEYDSYWWLGGLVICSLLTLIVIFYYLGLLCGVCGYDRHATPTTRGCVSNTGGVFLMVGVGLSFLFCWILMIIVVLTFVFGANVEKLICEPYISKELFRVLDTPYLLNEDWEYYLSGKLFNKSKMKLTFEQVYSDCKKNRGTYGTLHLQNSFNISEHLNINEHTGSISSELESLKVNLNIFLLGAAGRKNLQDFAACGIDRMNYDSYLAQTGKSPAGVNLLSFAYDLEAKANSLPPGNLRNSLKRDAQTIKTIHQQRVLPIEQSLSTLYQSVKILQRTGNGLLERVIRILASLDFAQNFITNNTSSVIIEETKKYGRTIIGYFEHYLQWIEFSISEKVASCKPVATALDTAVDVFLCSYIIDPLNLFWFGIGKATVFLLPALIFAVKLAKYYRRMDSEDVYDDVETIPMKNMENGNNGYHKDH VYGIHNPVMTSPSQH(SEQ ID NO: 1) The 3 extracellular domains of CD133 are underlined.Italics indicate signal sequence absent from mature, surface-expressedpolypeptide.

Example 2: Discovery and Characterization of Novel Antibody VariableRegions RW01 and RW03 Capable of Specifically Binding Human CD133

Cell Selections and Sequencing Data:

Cell-based selections for CD133-binding phage-Fab or phage-scFv wereperformed using the Cellectseq method as described in Example 1. Out of94 phage-Fab clones selected from the Library F output and subjected toELISA for determining their CD133-binding specificity, 77 clones werefound to bind to HEK293-CD133 cells at levels at least 1.5-fold higherthan to control HEK293 cells (FIG. 1A). In contrast, none of the clonesselected from the Library G output exhibited this CD133-bindingspecificity. The 94 clones selected from Library F were sequenced byamplifying DNA encoding the Fab variable regions (VL and VH regions)using M13-tagged sequencing primers. The Library F and Library G roundthree and four positive and negative outputs were sequenced via Illuminasequencing to identify binders enriched in the positive selection outputpools. The sequencing results indicated that 91 of the 94 clones werecomposed of clones each having one of 3 unique antibody variableregions, as represented by clones “phage-Fab RW03”, “phage-Fab C12” and“phage-Fab F5”. Specifically, out of the 94 clones, 89 clones shared thesame variable region sequences, 2 clones had unique variable regionsequences, and no sequence was obtained for 3 clones.

FIG. 1B shows representative cell-based ELISA results for binding toHEK293-CD133 and HEK293 cells by phage-Fab RW03 comprising “antibodyvariable region RW03”, phage-Fab C12 and phage-Fab F5. Only phage-FabRW03 showed preferential binding to HEK293-CD133 cells vs HEK293 cellsby a factor of at least 1.5. The Fab-encoding DNA sequences of phage-FabRW03, phage-Fab C12 and phage-Fab F5 were each cloned into anIPTG-inducible vector for protein expression and the expressed Fabs(“Fab RW03”, “Fab C12” and “Fab F5”, respectively) were purified fortesting via immunofluorescence (IF) assay, the results of which areshown in FIG. 2. The IF assay showed that Fab RW03 demonstrated highlyspecific binding to the HEK293-CD133 cells, with very little backgroundbinding to HEK293 cells, whereas Fab C12 and Fab F5 were found to bindnon-specifically to both HEK293-CD133 cells and HEK293 cells. Thespecific binding of Fab RW03 to HEK293-CD133 cells was also confirmed bycell-based ELISA (FIG. 3A). Furthermore, Fab RW03 also was found to bindto Caco-2 cells, which is a colorectal cancer cell line known to expressCD133 (FIG. 3B). These results were consistent with the Illuminasequencing results in which the variable region DNA sequences ofphage-Fab clone C12 and phage-Fab clone F5 appeared in both the positiveas well as the negative selection output pools whereas those ofphage-Fab RW03 appear only in the positive selection pool (data notshown). Additionally, the phage-Fab RW03 DNA sequence was the mostabundant sequence in both the round 3 and 4 output pools, which is alsoconsistent with the results of the cell-based ELISA in which the DNAsequences of 94% of the phage-Fab binders were those of phage-Fab RW03.

In addition to determining the DNA sequences encoding CD133-bindingphage-Fabs from Library F, the Illumina sequencing data from Library Gselection output pools was also analyzed to identify enriched binders(data not shown). DNA encoding the variable regions of 12 selectedphage-scFv clones was rescued via PCR amplification and used to generateexpression vectors for expression of 12 IgGs having variable regionscorresponding to those of the 12 selected phage-scFv clones. Of these 12IgGs, “lIgG RW01” containing “antibody variable region RW01” derivedfrom phage-scFv clone RW01 was validated for specifically binding toCD133+ cells by flow cytometry analysis. Similarly, “lIgG RW03” having“antibody variable region RW03” corresponding to that of Fab RW03 wasproduced and IgG RW01 and IgG RW03 were further tested in parallel.

The amino acid sequences of light chain (hK) and heavy chain of IgG RW01and IgG RW03, and of the heavy and light chain variable domains ofantibody variable regions RW01 and RW03 are shown in Table 2, and theamino acid sequences of complementarity-determining regions (CDRs) andheavy chain variable domain residues at positions 39, 55 and 66 ofantibody variable region RW01 comprised in IgG RW01 and of antibodyvariable region RW03 comprised in IgG RW03 are indicated in Table 3. Thenucleotide sequences encoding the CDRs and heavy chain variable domainresidues at positions 39, 55 and 66 of antibody variable region RW01comprised in IgG RW01 and of antibody variable region RW03 comprised inIgG RW03 are indicated in Table 4. The amino acid sequence of the lightchain variable domain of IgG RW01 corresponds to that of the Asp1 toLys106 segment of SEQ ID NO: 2; the amino acid sequence of the heavychain variable domain of IgG RW01 corresponds to that of the Glu1 toThr120 segment of SEQ ID NO: 3; the amino acid sequence of the lightchain variable domain of IgG RW03 corresponds to that of the Asp1 toLys109 segment of SEQ ID NO: 4; and the amino acid sequence of the heavychain variable domain of IgG RW03 corresponds to that of the Glu1 toSer118 segment of SEQ ID NO: 5.

TABLE 2 Amino acid sequences of light chain (hK) andheavy chain of IgG RW01 and IgG RW03, and ofheavy and light chain variable domains ofantibody variable regions RW01 and RW03.Light chain variable domain of antibody variable region RW01:DIQMTQSPSSLSASVGDRVTITCRASQGSSYVAWYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGVWSLITFGQG TKVEIK(SEQ ID NO: 28) Heavy chain variable domain of antibody variableregion RW01: EVQLVESGGGLVQPGGSLRLSCAASGFNTYYYGS

HWVRQAPGK GLEWVA

ISPYYGST

YADSVKGRFTISADTSKNTAYLQMNSL RAEDTAVYYCARHASSGYGHYAVYGIDYWGQGTLVTVSS(SEQ ID NO: 29) IgG RW01 - Light chain (hK) amino acid sequence:DIQMTQSPSSLSASVGDRVTITCRASQGSSYVAWYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGVWSLITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC(SEQ ID NO: 2) IgG RW01 - Heavy chain (hG1) amino acid sequence:EVQLVESGGGLVQPGGSLRLSCAASGFNIYYYGS

HWVRQAPG KGLEWVA

ISPYYGST

YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARHASSGYGHYAVYGIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSREDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK(SEQ ID NO: 3) Light chain variable domain of antibody variableregion RW03: DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSHAGHLFTF GQGTKVEIK(SEQ ID NO: 30) Heavy chain variable domain of antibody variableregion RW03: EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSS

HWVRQAPG KGLEWVA

IYPYYSYT

YADSVKGRFTISADTSKNTAYLQMNS LRAEDTAVYYCARFGSVAGFDYWGQGTLVTVSS(SEQ ID NO: 31) IgG RW03 - Light chain (hK) amino acid sequence:DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSHAGHLFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC(SEQ ID NO: 4) IgG RW03 - Heavy chain (hG1) amino acid sequence:EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSS

HWVRQAPG KGLEWVA

IYPYYSYT

YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFGSVAGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSREDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK(SEQ ID NO: 5) Underline identifies CDR sequence amino acid residues.Large font identifies FR amino acid residues at positions 39, 55 or 66randomized in the selection library used to identify antibody variableregions. Italics identifies immunoglobulin constant region amino acidresidues or nucleotide sequences encoding immunoglobulin constant regionamino acid residues.

TABLE 3 Amino acid sequences of CDRs and of FR residuesat heavy chain variable domain positions 39, 55and 66 of antibody variable region RW01 andantibody variable region RW03. Anti- Antibody Amino acid body segmentsequence IgG  CDR-L1 QGSSY RW01 (SEQ ID NO: 6) CDR-L2 SAS (SEQ ID NO: 7)CDR-L3 QQGVWSLIT (SEQ ID NO: 8) CDR-H1 GFNIYYYGS (SEQ ID NO: 9) CDR-H2ISPYYGST (SEQ ID NO: 10) CDR-H3 ARHASSGYGHYAVYGIDY (SEQ ID NO: 11)VH domain position 39 Met (adjacent to carboxy  terminal residue ofCDR-H1; FR2 residue) VH domain position 55 Ser (adjacent to amino terminal residue of CDR-H2; FR2 residue) VH domain position 66 Tyr(adjacent to carboxy  terminal residue of CDR-H2; FR3 residue)Segment spanning  GSMIYYYGS CDR-H1 (underlined) (SEQ ID NO: 32)and VH domain position 39 Segment spanning  SISPYYGSTYVH domain position (SEQ ID NO: 33) 55, CDR-H2  (underlined) and VHdomain position 66 IgG  CDR-L1 QSVSSA RW03 (SEQ ID NO: 12) CDR-L2 SAS(SEQ ID NO: 13) CDR-L3 QQYSHAGHLFT (SEQ ID NO: 14) CDR-H1 GFNLSSSS(SEQ ID NO: 15) CDR-H2 IYPYYSYT (SEQ ID NO: 16) CDR-H3 ARFGSVAGFDY(SEQ ID NO: 17) VH domain position 39 Ile (adjacent to carboxy terminal residue of CDR-H1; FR2 residue) VH domain position 55 Tyr(adjacent to amino  terminal residue of CDR-H2; FR2 residue)VH domain position 66 Tyr (adjacent to carboxy  terminal residue ofCDR-H2; FR3 residue) Segment spanning  GFNLSSSSI CDR-H1 (underlined)(SEQ ID NO: 34) and VH domain position 39 Segment spanning  YIYPYYSYTYVH domain position (SEQ ID NO: 35) 55, CDR-H2  (underlined) and VHdomain position 66

TABLE 4 Nucleotide sequences encoding complementarity-determining regions (CDRs) and FR residues atheavy chain variable domain positions 39, 55and 66 of antibody variable region RW01 andantibody variable region RW03. Anti- Antibody body segmentNucleotide sequence IgG CDR-L1 CAGGGTTCTTCTTAC RW01 (SEQ ID NO: 36)CDR-L2 TCTGCATCC (SEQ ID NO: 37) CDR-L3 CAGCAAGGTGTTTGGTCTCTGATCACG(SEQ ID NO: 38) CDR-H1 GGCTTCAACATCTACTACTACGGTTCT (SEQ ID NO: 39)CDR-H2 ATTTCTCCTTACTACGGCTCTACT (SEQ ID NO: 40) CDR-H3GCTCGCCATGCTTCTTCTGGTTACGGTCATTAC GCTGTTTACGGTATTGACTAC (SEQ ID NO: 41)VH domain ATG position  39 (adja- cent to carboxy terminal residue of CDR- H1; FR2 residue) VH domain TCT position  55 (adja- cent to aminoterminal residue  of CDR- H2; FR2 residue) VH domain TAC position 66 (adja- cent to carboxy terminal residue  of CDR- H2; FR3 residue)Segment GGCTTCAACATCTACTACTACGGTTCTATG spanning (SEQ ID NO: 42) CDR-H1(under- lined) and VH domain position  39 SegmentTCTATTTCTCCTTACTACGGCTCTACTTAC spanning  (SEQ ID NO: 43) VH domainposition  55, CDR-H2 (under- lined) and VH domain position  66 IgGCDR-L1 CAGTCCGTGTCCAGCGCT RW03 (SEQ ID NO: 44) CDR-L2 TCGGCATCC(SEQ ID NO: 45) CDR-L3 CAGCAATACTCTCATGCTGGTCATCTGTTCACG (SEQ ID NO: 46)CDR-H1 GGCTTCAACCTCTCTTCTTCTTCT (SEQ ID NO: 47) CDR-H2ATTTATCCTTATTATAGCTATACT (SEQ ID NO: 48) CDR-H3GCTCGCTTCGGTTCTGTTGCTGGTTTTGACTAC (SEQ ID NO: 49) VH domain ATCposition  39 (adja- cent to carboxy terminal residue  of CDR- H1; FR2residue) VH domain TAT position  55 (adja- cent to amino terminalresidue  of CDR- H2; FR2 residue) VH domain TAT position  66 (adja-cent to carboxy terminal residue  of CDR- H2; FR3 residue) SegmentGGCTTCAACCTCTCTTCTTCTTCTATC spanning (SEQ ID NO: 50) CDR-H1 (under-lined) and VH domain position  39 Segment TATATTTATCCTTATTATAGCTATACTTATspanning  (SEQ ID NO: 51) VH domain position  55, CDR-H2 (under- lined)and VH domain position  66

The nucleotide sequences of DNA encoding light chain (hK) and heavychain of IgG RW01 and IgG RW03, and the heavy chain and light chainvariable domains of antibody variable regions RW01 and RW03 are shown inTable 5.

TABLE 5 Nucleotide sequences of DNA encoding light chain(hK) and heavy chain of IgG RW01 and IgG RW03,and heavy chain and light chain variable domainsof antibody variable regions RW01 and RW03.Light chain variable domain of antibody variable region RW01:5′-GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGGTTCTTCTTACGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCTGCATCCTACCTCTACTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAGGTGTTTGGTCTCTGATCACGTTCGGACAGGGTACCAAGGTGGAGATCAAA-3′ (SEQ ID NO: 52)Heavy chain variable domain of antibody variable region RW01:5′-GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACCTCTCTTCTTCTT CT

CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGA ATGGGTTGCA

ATTTATCCTTATTATAGCTATACT

TATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTTCGGTTCTGTTGCTGGTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG-3′ (SEQ ID NO: 53)IgG RW01 - Light chain (hK) nucleotide sequence:5′-GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGGTTCTTCTTACGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCTGCATCCTACCTCTACTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAGGTGTTTGGTCTCTGATCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT-3′ (SEQ ID NO: 18)IgG RW01 - Heavy chain (hG1) nucleotide sequence:5′-GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATCTACTACTACG GTTCT

CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCT GGAATGGGTTGCA

ATTTCTCCTTACTACGGCTCTACT

ATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCCATGCTTCTTCTGGTTACGGTCATTACGCTGTTTACGGTATTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAA A-3′ (SEQ ID NO: 19)Light chain variable domain of antibody variable region RW03:5′-GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATCCAGCCTCTACTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAATACTCTCATGCTGGTCATCTGTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAA-3′ (SEQ ID NO: 54)Heavy chain variable domain of antibody variable region RW03:5′-GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACCTCTCTTCTTCTT CT

CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTG GAATGGGTTGCA

ATTTATCCTTATTATAGCTATAC T

TATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTTCGGTTCTGTTGCTGGTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG-3′ (SEQ ID NO: 55)IgG RW03 - Light chain (hK) nucleotide sequence:5′-GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATCCAGCCTCTACTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAATACTCTCATGCTGGTCATCTGTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGT-3′(SEQ ID NO: 20) IgG RW03 - Heavy chain (hG1) nucleotide sequence:5′-GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACCTCTCTTCTTCTT CT

CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTG GAATGGGTTGCA

ATTTATCCTTATTATAGCTATAC T

TATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTTCGGTTCTGTTGCTGGTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA-3′ (SEQ ID NO: 21) Underlineidentifies nucleotide sequences encoding CDR amino acid residues.Large-font identifies nucleotide sequences encoding FR amino acidresidues at positions 39, 55 and 66 randomized in the selection libraryused to identify antibody variable regions. Italics identify nucleotidesequences encoding immunoglobulin constant region amino acid residues.

Example 3: IgG RW01 and IgG RW03 Bind CD133 Expressed at the CellSurface with Approximately Single-Digit Nanomolar and SubnanomolarAffinity, Respectively

The IgG RW01 and IgG RW03 antibodies were tested via flow cytometry fortheir capacity to bind HEK293-CD133 cells, after which half maximalbinding concentrations of each antibody to the cells were estimated.Specifically, HEK293-CD133 cells were incubated with serial dilutions ofeach antibody, binding was detected with an anti-human Fab′2 secondaryantibody and the data was fitted to a line of best fit using the SigmaPlot graphing program. FIG. 4 shows the EC50 curve for IgG RW01, whichhad a calculated EC50 of 2.5 nM and the curve for IgG RW03, which had acalculated EC50 of 0.5 nM.

Example 4: IgG RW01 and IgG RW03 can be Used to Specifically Bind andDetect Cell Surface CD133 in Pancreatic Cancer Cells and ColorectalCancer Cells, as Shown Via Flow Cytometry Analysis

The IgG RW01 and IgG RW03 antibodies were assessed via flow cytometryfor binding to the following cancer cell lines: Caco-2, a colon cancercell line known to express CD133; the pancreatic cancer cell lines HPAC,PL45, RWP-1 and SU8686; and the ovarian carcinoma cell line Ovcar-8.HEK293 and HEK293-CD133 cells were used as negative and positivecontrols, respectively. As shown in FIG. 5, both antibodies, at 5 μg/ml,bind CD133 at the surface of HEK293-CD133 control cells, at the surfaceof Caco-2 colorectal cancer cells and at the surface of HPAC, PL45 andRWP-1 pancreatic cancer cells. Bimodal staining peaks are observed inHPAC and PL45 cells and broader peaks such as those observed for theengineered cell line HEK293-CD133 are most likely a result of aheterogeneously expressing population of cells, contrasted with thenarrow peak observed with RWP-1 cells indicating a more homogeneouslyexpressing cell population.

Example 5: IgG RW01 and IgG RW03 can be Used to Specifically Bind,Detect and Subcellularly Localize Cellular CD133, as Shown ViaImmunofluorescence Analysis

The IgG RW01 and IgG RW03 antibodies were tested for binding tocell-expressed CD133 in an immunofluorescence assay (FIG. 6). The assayshows that IgG RW01 and IgG RW03 bind to HEK293-CD133 cells (indicatingcellular sublocalization of CD133), but not to HEK293 cells.

Example 6: IgG RW01 and IgG RW03 can Each be Used to Detect DenaturedCD133 in Whole Cell Lysate of Colorectal Cancer Cells, as Shown ViaWestern Blot Analysis

Whole cell lysates of Caco-2 colorectal cancer cells were prepared andthe capacity of IgG RW01 and IgG RW03 to detect denatured CD133 in thelysate was analyzed via Western blotting assay. HEK293-CD133 cells andHEK293 cells were used as positive and negative controls, respectively.FIG. 7 shows that IgG RW01 and IgG RW03 each detect denatured CD133 inlysate of Caco-2 cells and HEK293-CD133 cells, which are bothCD133-positive, but not in that of HEK293 cells.

Example 7: Epitopes—Fab RW01 and Fab RW03 do not Compete with IgG RW03and IgG RW01, Respectively, for Binding to CD133

Experiments were performed to determine whether antibody variable regionRW01 and antibody variable region RW03 compete with each other forbinding to cell-expressed CD133. Cells were initially incubated with a25 nM concentration of either Fab RW01 or Fab RW03, and serial dilutionsof IgG RW03 or IgG RW01, respectively, were added, and the IgG bindingwas detected using a secondary antibody against human Fc. The resultsfor each antibody can be seen in FIGS. 8 (a) and (b) and indicate thatIgG RW01 can bind the cells in the presence of saturating RW03 Fab andIgG RW03 can bind in the presence of RW01 Fab.

Example 8: Treatment with IgG RW01 or IgG RW03 can be Used toSignificantly Reduce Total Cellular CD133 Protein Levels in Caco-2Colorectal Cancer Cells

To investigate the effect of IgG RW01 and IgG RW03 on CD133 proteinlevels in cancer cells, Caco-2 colorectal cancer cells were incubatedwith either IgG RW01 or IgG RW03 for 24 hours at 37° C., and whole celllysates were analyzed by Western blot using AC133 anti-CD133 antibody(Miltenyi Biotec) as probe and anti-human IgG (H+L) antibody (JacksonImmunoResearch) as negative antibody control. As FIG. 9 shows, treatmentwith RW01 IgG or RW03 IgG significantly reduced CD133 protein levels, ascompared to the untreated and anti-human IgG control treatments. Toassess the effect of the observed reduction in CD133 protein levels onWnt signaling in the cells, β-catenin levels were also analyzed, howeverthere were no observed differences on β-catenin protein stabilitybetween control antibody-treated and IgG RW01- or IgG RW03-treatedsamples.

Example 9: Single-Chain Fab RW03 can be Used for Targeting Bispecific TCell Engagers (BiTEs) to CD133-Positive Cells

Vectors were constructed for expression of four novelanti-CD133×anti-CD3 bi-specific T-cell engagers (BiTEs; FIG. 10A). Eachof the four BiTEs incorporates an anti-CD3 scFv, and furtherincorporates either anti-CD133 Fab RW03 (Fab-based BiTEs) or ananti-CD133 single-chain Fab (scFab) incorporating the variable regionsof Fab RW03 (“scFab RW03”; scFab-based BiTEs). In each of the 4 BiTEs,the Fab or scFab is linked to the VH domain of the scFv. The 4 BiTEsinclude two Fab-based variants, in which the scFv is linked either tothe light chain (“BiTE #1”) or heavy chain-derived portion (“BiTE #2”)of the Fab. The other two BiTEs are scFab-based variants, in which thescFv is similarly linked either to the heavy chain-derived segment(“BiTE #3”) or light chain segment (“BiTE #4”) of the scFab.

The amino acid sequences of BiTE #1, BiTE #2, BiTE #3 and BiTE #4 areshown in Table 6.

TABLE 6 Configurations and amino acid sequences of BiTE#1, BiTE #2, BiTE #3 and BiTE #4 polypeptides. BiTE #1 Configuration:VL-CL (RW03)-(G4S)-VH-(G4S)3-VL (CD3) VH-CH (RW03)Amino acid sequence of VL-CL (RW03)-(G4S)-VH- (G4S)3-VL:DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSHAGHLFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPL TFGAGTKLELK(SEQ ID NO: 22) Amino acid sequence of VH-CH (RW03):EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSSIHWVRQAPGKGLEWVAYIYPYYSYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFGSVAGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVIQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 23) BiTE #2 Configuration:VH-CH (RW03)-(G4S)-VH-(G4S)3-VL (CD3) VL-CL (RW03)Amino acid sequence of VH-CH (RW03)-(G4S)-VH- (G4S)3-VL (CD3):EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSSIHWVRQAPGKGLEWVAYIYPYYSYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFGSVAGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRESGSGSGTSYSLTISSMEAEDAATY YCQQWSSNPLTFGAGTKLELK(SEQ ID NO: 24) Amino acid sequence of VL-CL (RW03):DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRESGSRSGTDFTLTISSLQPEDFATYYCQQYSHAGHLFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC(SEQ ID NO: 25) BiTE #3 Configuration:VL-CL-linker-VH-CH (RW03)-(G4S)-VH-(G4S)3-VL (CD3) Amino acid sequence:DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRESGSRSGTDFTLTISSLQPEDFATYYCQQYSHAGHLFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGEVQLVESGGGLVQPGGSLRLSCAASGFNLSSSSIHWVRQAPGKGLEWVAYIYPYYSYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFGSVAGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTETRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRESGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 26) BiTE #4 Configuration:VH-CH-linker-VL-CL (RW03)-(G4S)-VH-(G4S)3-VL (CD3) Amino acid sequence:EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSSIHWVRQAPGKGLEWVAYYIYPYYSYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFGSVAGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYSHAGHLFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 27) Underline identifies CDRsequence. Italics represent immunoglobulin constant region amino acids.Linker amino acids indicated in bold

HEK293 cells transiently transfected with the BiTE-expression vectorswere capable of readily expressing BiTE #1, BiTE #2, BiTE #3 and BiTE #4expressed and purified from, as shown via Western blot analysis ofreduced and non-reduced whole cell lysates of transfectants (FIG. 10B).

BiTE #1, BiTE #2, BiTE #3 and BiTE #4 have the Capacity to Bind to CellSurface CD133 and CD3

The capacity of the purified BiTEs to specifically bind cell surfaceCD133 was determined using flow cytometry analysis of staining ofHEK293-CD133 cells vs HEK293 cells with the BiTEs. As shown in FIG. 11Aand FIG. 11B, BiTE #1, BiTE #2, BiTE #3 and BiTE #4 each binds toHEK293-CD133 cells significantly more than to HEK293 cells, even atconcentrations as low as 0.073-0.11 microgram/ml. The purified BiTEswere further tested in an ELISA to determine their capacity to bind toCD3 in the form of CD3 epsilon/gamma and CD3 epsilon/delta. As shown inFIG. 12, the purified BiTEs bind to CD3 as well as the positive controlantibodies (UCHT1, OKT3). These BiTEs can bind to both CD133 and CD3.

Example 10: Human CD133-Specific Chimeric Antigen Receptor (CAR)Modified T Cells Target Patient-Derived Glioblastoma Brain Tumors

A single chain variable fragment (scFv) was derived from RW03 (describedabove) and a second-generation CAR was generated. Anti-CD133 scFv with amyc tag was cloned in frame with a human CD8 leader sequence, CD8atransmembrane domain, CD28, and hCD3 (human CD3zeta) signaling tail inthe lentiviral construct pCCL-ΔNGFR vector in two differentorientations: Light chain-linker-Heavy chain (CD133 CAR-LH) and Heavychain-linker-Light chain (CD133 CAR-HL).

Following lentiviral preparation, T cells isolated from PBMCs weretransduced with CD133 CAR-LH and CD133 CAR-VH constructs. Aftersuccessful T cell engineering, the expression of ΔNGFR and myc tag wasanalyzed using flow cytometry to confirm the efficiency of transductionand surface expression of anti-CD133 respectively. While expression ofΔNGFR was observed in all CAR T cells (including controls), expressionof the c-myc tag in both variations of CARs, CD133 CAR-HL and CD133CAR-LH was found. Furthermore, Presto Blue-based killing assays wereused to test the ability of CD133 CARs to selectively bind and killCD133+ GBM brain tumor initiating cell lines (BTICs). CD133-specificCAR-T cells were cytotoxic to CD133+ GBMs. Co-culturing CD133 CAR-Tcells with GBMs triggered T cell activation and proliferation,validating this adoptive T-cell therapeutic strategy.

Human T cells expressing CD133-specific CARs were engineered by cloningscFv comprising the heavy and light chain variable domains of antibodyvariable region RW03 (disclosed above), a short marker epitope of c-myc,the hinge region from murine CD8 and the transmembrane and cytoplasmicportions of murine CD28 and CD3, (FIG. 13). The human CD133-CAR wascloned into the lentiviral transfer vector pCCL-ΔNGFR and packaged aslentiviruses. The c-myc tag in the extracellular domain was used tovalidate CAR expression. ΔNGFR was used as a cell-surface marker fortracking and sorting of transduced cells.

Transduced T cells consisted of CD4-positive and CD8-positive cells withboth subsets expressing CD133-specific CARs (FIG. 14). After successfultransduction, the expression of ΔNGFR (CD271) was observed in all CAR Tcells (including controls), however increased expression of the c-myctag was found in the CD133 CAR-HL and CD133 CAR-LH cells only (FIG. 15).

CD133-specific CAR-T cells were incubated with GBM BT459, stained andanalyzed for surface expression of T cell activation markers CD69 andCD25 after 18 hours (FIG. 16). Both, CD4+(T-helper) andCD8+(T-cytotoxic) cells showed upregulation in surface expression levelsof activation markers. The elevated expression was detected only in thepresence of CD133-specific CAR and not CAR-T control.

CD133-specific CAR T-cells showed enhanced proliferation capabilityafter being co-cultured with CD133^(high) GBM cells (FIG. 17, top row).CD133-specific CAR T cells specifically recognized and kill tumor cellsin CD133^(high) GBMs and medulloblastoma (Control), while having noeffect on CD133^(low) GBM cells (FIG. 17, bottom row). CD133^(high) andCD133^(low) GBMs were defined based on the percentage of CD133-positivecells present in the tumor cell culture. GBM cultures with >90% CD133+cells were defined as CD133^(high) GBMs and cultures with <5% CD133+cells were defined as CD133^(b0)w GBMs.

Tumor-engrafted mice were injected i.e. with CAR-CON (control) T cells(FIG. 18A) and with CAR-CD133 T cells at an effector to target ratio(E:T)=2:1 (FIG. 18B). Tumors formed in mouse brain intracraniallytreated with CAR-CD133 T cells were significantly less aggressive andinvasive compared to control (as assessed by COX IV staining for humancells) (n=4). Mouse xenografts generated after CAR-CD133 T celltreatment had significant less tumor mass (FIG. 18C).

Treatment of GBM tumor-bearing mice with CD133-specific CAR-T cellsyielded extended survival in mice and significant reductions in braintumor burden.

Example 11. Preclinical Validation of a Novel CD133/CD3 BispecificT-Cell Engager (BiTE) Antibody to Target Patient-Derived GlioblastomaCells

The BiTE format has been evaluated against a variety of tumor-associatedantigens, including CD19, CD20, EpCAM, EGFR, MUC-1, CEA, CD133, EphA2and HER2/neu (reviewed in Baeuerle et al. 2009). Clinical trialsinvestigating BiTEs include Blinatumomab for leukemia patients(NCT00274742), AMG110/MT110 for lung/colorectal/breast/ovarian cancerpatients (NCT00635596), AMG211/MED1565 for gastrointestinaladenocarcinoma patients (NCT01284231) and AMG212/BAY2010112 for prostatecancer patients (NCT0173475). BiTEs exhibiting specificity for two GBMtumor cell surface antigens CD133 (Prasad et al, 2015) and EGFRvIII(Choi et al, 2013) have also been shown to be induce anti-tumorigenicactivity in xenograft tumor models. Importantly, preclinical evaluationof EGFRvIII-specific BiTEs delivered intravenously showed tumorreduction/shrinkage, extending survival in mice with well-establishedEGFRvIII-expressing GBM.

As described in Example 9, CD133-specific BiTEs or RW03×CD3 wereconstructed that consist of two arms; one arm recognizes the tumorantigen (CD133) while the second is specific to CD3 antigen. The BiTEswere constructed in four different conformations and dual bindingspecificity was confirmed using flow cytometry. Using CD133^(high) andCD133^(low) primary GBM lines, the binding of BiTEs to CD133+ cells wasvalidated. Further analysis showed binding of BiTEs to human T cellsknown to express CD3 within a population of healthy donor peripheralblood mononuclear cells. BiTEs redirecting T cells to kill GBMs wereobserved, with greater efficiency observed in CD133^(high) GBMs,validating BiTE target specificity.

Specifically, using CellectSeq, a novel methodology that combines use ofphage-displayed synthetic antibody libraries and high-throughput DNAsequencing technology, CD133-specific monoclonal antibody ‘RW03’ wasdeveloped (FIG. 19A). Following validation of CD133 RW03 antibody,CD133-specific BiTEs or RW03×CD3 was constructed that had two arms; onearm recognizes the tumor antigen (CD133) while the second is specific toCD3 antigen. The BiTEs were constructed in four different conformations(FIG. 19B).

Flow cytometry was used to confirm dual specificity of CD133×CD3 BiTEsagainst cells expressing the appropriate antigen. CD133×CD3 BiTEs bindto GBM tumor expressing CD133 in similar capacity when compared withbinding to commercially available CD133 (Miltenyi) monoclonal antibody(FIG. 20A). Analyses revealed binding of CD133×CD3 BiTEs to human Tcells known to express CD3 within a population of healthy donorperipheral blood mononuclear cells (PBMCs) (FIG. 20B).

T cells incubated with BiTEs and CD133^(high) GBM BT459 were stained andanalyzed for surface expression of activation markers CD69 and CD25(FIG. 21). Both, CD4+(T-helper) and CD8+(T-cytotoxic) cells showedupregulation in surface expression levels of the activation markers. Theelevated expression was detected only in the presence of CD133 BiTE inthe co-culture.

GBM cells form a monolayer when plated alone (FIG. 22A(a) or plated withT cells (FIG. 22A(b)); however, addition of BiTEs (FIG. 22A, c and d)recruits T cells to GBM cells forming spherical clusters. Addition ofBiTEs to GBM cells co-cultured with T cells leads to significant celldeath of GBM cells as determined through alamar-blue cytotoxicity assay(FIG. 22B). Quantification of tumor cell lysis by flow cytometry-basedLive-Dead staining (with IR dye) (FIG. 22C). Tumor cells (CFSE-labeled)and T cells (E:T ratio, 1:2) in presence and absence of CD133 BiTEsafter 24 hours show BiTE-mediated GBM cell death.

NSG (NOD scid gamma) mice were implanted i.c. with CD133^(high) GBMcells and upon successful engraftment, were treated with unstimulatedPBMCs with or without BiTEs (Total 4 doses over 2 weeks) (FIG. 23).Tumors formed in mouse brain intracranially treated with CD133 BiTEswere significantly less aggressive and invasive compared to control (asassessed by COX IV staining for human cells) (n=4) (FIGS. 23, A and B)).Mouse xenografts generated after BiTE treatment had less tumor burden(FIG. 23C) and maintained a significant survival advantage over controlmice (n=7) (FIG. 23D).

Methods for Examples 10 and 11

Flow Cytometric Characterization:

Patient-derived GBM lines were dissociated and single cells resuspendedin PBS+2 mM EDTA. Cell suspensions were stained with anti-CD133,anti-CD69, anti-CD25, anti-CD8, anti-CD4 or matched isotype controls(Miltenyi/BD Biosciences). Samples were run on a MoFlo XDP Cell Sorter(Beckman Coulter). Dead cells were excluded using the viability dye7AAD. (1:10; Beckman Coulter). Compensation was performed using mouseIgG CompBeads (BD). Surface marker expression was defined as positive ornegative based on the analysis regions established using the isotypecontrol.

Cell Proliferation Assay:

Single cells were plated in a 96-well plate at a density of 1,000cells/200 μL per well in quadruplicate and incubated for four days. 20μL of Presto Blue (Invitrogen) was added to each well approximately 2 hprior to the readout time point. Fluorescence was measured using aFLUOstar Omega Fluorescence 556 Microplate reader (BMG LABTECH) atexcitation and emission wavelengths of 535 nm and 600 nm, respectively.Readings were analyzed using Omega analysis software.

Quantitative Cytotoxicity Assays:

Prestoblue killing and LDH cytotoxicity assays with differenteffector/target (E:T) ratios was performed to determine the efficiencyof BiTEs to redirect T cells and kill CD133-expressing GBM BTICs.

In Vivo GBM Intracranial Injections and H&E Staining of XenograftTumors:

All experimental procedures involving animals were reviewed and approvedby McMaster University Animal Research Ethics Board (AREB). NOD-SCIDmice were used for all experiments. Mice were anaesthesized using gasanaesthesia (Isoflurane: 5% induction, 2.5% maintenance) identifiedusing ear notches following minimally invasive surgery, and monitoredfor recovery. Intracranial injections were performed as previouslydescribed (1). Briefly, 10 μL of cell suspension was injected into theright frontal lobe of 8-10 week old mice. Mice were monitored weekly forsigns of illness, and upon reaching endpoint, brains and lungs (for ITand ICa injections) were harvested, sectioned, and paraffin-embedded forhematoxylin and eosin (H&E). Images were scanned using an Aperio SlideScanner and analyzed by ImageScope v11.1.2.760 software (Aperio).

While the present application has been described with reference toexamples, it is to be understood that the scope of the claims should notbe limited by the embodiments set forth in the examples, but should begiven the broadest interpretation consistent with the description as awhole.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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The invention claimed is:
 1. An antibody or an antigen binding fragmentthereof that specifically binds to a CD133 polypeptide, wherein saidantibody or antigen binding fragment thereof comprises a light chainvariable domain comprising complementarity determining regions (CDRs)comprising SEQ ID NOs: 12, 13, and 14 and a heavy chain variable domaincomprising CDRs comprising SEQ ID NOs: 15, 16, and
 17. 2. The antibodyor antigen binding fragment thereof according to claim 1, wherein saidantibody is a single chain variable fragment (scFv) antibody or a singlechain Fab fragment (scFab) antibody.
 3. The antibody or antigen bindingfragment thereof according to claim 1, wherein said antigen bindingfragment is selected from a Fab fragment, a Fab′ fragment, and an Fvfragment.
 4. The antibody or antigen binding fragment thereof accordingto claim 1, wherein said antibody is a humanized antibody or a chimericantibody.
 5. The antibody or antigen binding fragment thereof accordingto claim 1, wherein the light chain variable domain comprises an aminoacid sequence that is at least 70% identical to SEQ ID NO: 30 and/orwherein the heavy chain variable domain comprises an amino acid sequencethat is at least 70% identical to SEQ ID NO:
 31. 6. The antibody orantigen binding fragment thereof according to claim 1, wherein the lightchain variable domain comprises the amino acid sequence of SEQ ID NO: 30and/or wherein the heavy chain variable domain comprises the amino acidsequence of SEQ ID NO:
 31. 7. The antibody or antigen binding fragmentthereof according to claim 1, wherein the CD133 polypeptide is a humanCD133 polypeptide.
 8. The antibody or antigen binding fragment thereofaccording to claim 7, wherein the human CD133 polypeptide comprises SEQID NO:
 1. 9. The antibody or antigen binding fragment thereof accordingto claim 1, wherein the CD133 polypeptide is denatured.
 10. The antibodyor antigen binding fragment thereof according to claim 1, wherein theCD133 polypeptide is on the surface of a cell.
 11. The antibody orantigen binding fragment thereof according to claim 1, wherein saidantibody or antigen binding fragment thereof comprises human constantregions.
 12. The antibody or antigen binding fragment thereof accordingto claim 1, wherein said antibody is an IgG antibody.
 13. The antibodyor antigen binding fragment thereof according to claim 1, wherein saidantibody is an IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, IgE or IgMantibody.
 14. The antibody or antigen binding fragment thereof accordingto claim 1, wherein said antibody is a bispecific antibody.
 15. Theantibody or antigen binding fragment thereof according to claim 14,wherein said bispecific antibody binds to a CD3 polypeptide.
 16. Theantibody or antigen binding fragment thereof according to claim 14,wherein said bispecific antibody comprises: (a) a polypeptide comprisingSEQ ID NO: 22 and a polypeptide comprising SEQ ID NO: 23; (b) apolypeptide comprising SEQ ID NO: 24 and a polypeptide comprising SEQ IDNO: 25; (c) a polypeptide comprising SEQ ID NO: 26; or (d) a polypeptidecomprising SEQ ID NO:
 27. 17. A chimeric antigen receptor (CAR) thatspecifically binds to a CD133 polypeptide, wherein said CAR comprises anextracellular antigen binding domain comprising the antibody or antigenbinding fragment thereof according to claim
 1. 18. The antibody orantigen binding fragment thereof according to claim 1, wherein saidantibody or antigen binding fragment thereof is detectably labeled. 19.An immunoconjugate comprising the antibody or antigen binding fragmentthereof according to claim 1 and an effector agent.
 20. Theimmunoconjugate according to claim 19, wherein said effector agent is ananti-neoplastic agent or a toxin.
 21. A pharmaceutical compositioncomprising the antibody or antigen binding fragment thereof according toclaim 1 and a carrier.
 22. A method for treating cancer expressing CD133in a subject, said method comprising administering to the subject aneffective amount of the antibody or antigen binding fragment thereofaccording to claim
 1. 23. The method according to claim 22, wherein saidcancer is selected from a metastatic melanoma, a brain cancer, aprostate cancer, a pancreatic cancer, and a colon cancer.
 24. The methodaccording to claim 23, wherein said brain cancer is a glioblastoma or amedulloblastoma.
 25. A method for treating cancer expressing CD133 in asubject, said method comprising administering to the subject aneffective amount of the bispecific antibody according to claim
 15. 26. Amethod for treating cancer expressing CD133 in a subject, said methodcomprising administering to the subject an effective amount of T cellsexpressing a CAR according to claim
 17. 27. A method for detecting CD133and/or quantifying the levels of CD133 expressed by cells, said methodcomprising contacting a sample suspected of comprising CD133 or cellsexpressing CD133 with the antibody or antigen binding fragment thereofaccording to claim 1.